No novel coronaviruses identified in a large collection of human nasopharyngeal specimens using family-wide CODEHOP-based primers.

Novel viruses might be responsible for numerous disease cases with unknown etiology. In this study, we screened 1800 nasopharyngeal samples from adult outpatients with respiratory disease symptoms and healthy individuals. We employed a reverse transcription (RT)-PCR assay and CODEHOP-based primers (CT12-mCODEHOP) previously developed to recognize known and unknown corona- and toroviruses. The CT12-mCODEHOP assay detected 42.0 % (29/69) of samples positive for human coronaviruses (HCoV), including HCoV-229 (1/16), HCoV-NL63 (9/17), and HCoV-OC43 (19/36), and additionally HCoV-HKU1 (3), which was not targeted by the diagnostic real-time PCR assays. No other coronaviruses were identified in the analyzed samples.

Organ tropism of murine coronavirus does not correlate with the expression levels of the membrane-anchored or secreted isoforms of the carcinoembryonic antigen-related cell adhesion molecule 1 receptor.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is the sole known receptor of murine hepatitis virus (MHV) A59, but the available, often qualitative, data about CEACAM1 expression does not explain MHV organ tropism. Ceacam1 transcripts undergo alternative splicing resulting in multiple isoforms, including secreted CEACAM1 isoforms that can neutralize the virus. We determined the quantities of Ceacam1 transcripts encoding membrane-bound and secreted isoforms in mouse organs and a set of cell lines. In vivo, the lowest receptor mRNA levels were found in brain and muscle and these were similar to those in easily infectable cultured cells. While the quantities of the receptor transcripts varied between mouse organs, their abundance did not correlate with susceptibility to MHV infection. The proportion of transcripts encoding secreted isoforms also could not explain the selection of sites for virus replication, as it was constant in all organs. Our data suggest that neither of the two CEACAM1 isoforms defines MHV organ tropism.

An RNA pseudoknot is required for production of yellow fever virus subgenomic RNA by the host nuclease XRN1.

Cells and mice infected with arthropod-borne flaviviruses produce a small subgenomic RNA that is colinear with the distal part of the viral 3'-untranslated region (UTR). This small subgenomic flavivirus RNA (sfRNA) results from the incomplete degradation of the viral genome by the host 5'-3' exonuclease XRN1. Production of the sfRNA is important for the pathogenicity of the virus. This study not only presents a detailed description of the yellow fever virus (YFV) sfRNA but, more importantly, describes for the first time the molecular characteristics of the stalling site for XRN1 in the flavivirus genome. Similar to the case for West Nile virus, the YFV sfRNA was produced by XRN1. However, in contrast to the case for other arthropod-borne flaviviruses, not one but two sfRNAs were detected in YFV-infected mammalian cells. The smaller of these two sfRNAs was not observed in infected mosquito cells. The larger sfRNA could also be produced in vitro by incubation with purified XRN1. These two YFV sfRNAs formed a 5'-nested set. The 5' ends of the YFV sfRNAs were found to be just upstream of the previously predicted RNA pseudoknot PSK3. RNA structure probing and mutagenesis studies provided strong evidence that this pseudoknot structure was formed and served as the molecular signal to stall XRN1. The sequence involved in PSK3 formation was cloned into the Sinrep5 expression vector and shown to direct the production of an sfRNA-like RNA. These results underscore the importance of the RNA pseudoknot in stalling XRN1 and also demonstrate that it is the sole viral requirement for sfRNA production.

Characterization of hepatitis C virus NS3 modifications in the context of replication.

Post-translational modifications (PTMs) of viral proteins regulate various stages of infection. With only 10 proteins, hepatitis C virus (HCV) can orchestrate its complete viral life cycle. HCV non-structural protein 3 (NS3) has many functions. It has protease and helicase activities, interacts with several host-cell proteins and plays a role in translation, replication and virus-particle formation. Organization of all these functions is necessary and could be regulated by PTMs. We therefore searched for modifications of the NS3 protein in the subgenomic HCV replicon. When performing a tag-capture approach coupled with two-dimensional gel electrophoresis analyses, we observed that isolated His6-NS3 yielded multiple spots. Individual protein spots were digested in gel and analysed by mass spectrometry. Differences observed between the individual peptide mass fingerprints suggested the presence of modified peptides and allowed us to identify N-terminal acetylation and an adaptive mutation of NS3 (Q1067R). Further analysis of other NS3 variants revealed phosphorylation of NS3.

Intracellular restriction of a productive noncytopathic coronavirus infection.

Virus infection in vitro can either result in a cytopathic effect (CPE) or proceed without visible changes in infected cells (noncytopathic infection). We are interested in understanding the mechanisms controlling the impact of coronavirus infection on host cells. To this end, we compared a productive, noncytopathic infection of murine hepatitis virus (MHV) strain A59 in the fibroblastlike cell line NIH 3T3 with cytopathic MHV infections. Infected NIH 3T3 cells could be cultured for up to 4 weeks without apparent CPE and yet produce virus at 10(7) to 10(8) PFU/ml. Using flow cytometry, we demonstrated that NIH 3T3 cells expressed as much MHV receptor CEACAM1 as other cell lines which die from MHV infection. In contrast, using quantitative reverse transcription-PCR and metabolic labeling of RNA, we found that the rate of viral RNA amplification in NIH 3T3 cells was lower than the rate in cells in which MHV induces a CPE. The rate of cellular RNA synthesis in contact-inhibited confluent NIH 3T3 cells was also lower than in cells permissive to cytopathic MHV infection. However, the induction of cellular RNA synthesis in growing NIH 3T3 cells did not result in an increase of either viral RNA amplification or CPE. Our results suggest that a specific, receptor CEACAM1-independent mechanism restricting coronaviral RNA synthesis and CPE is present in NIH 3T3 and, possibly, other cells with preserved contact inhibition.

Hepatitis C virus NS4B carboxy terminal domain is a membrane binding domain.

BACKGROUND: Hepatitis C virus (HCV) induces membrane rearrangements during replication. All HCV proteins are associated to membranes, pointing out the importance of membranes for HCV. Non structural protein 4B (NS4B) has been reported to induce cellular membrane alterations like the membranous web. Four transmembrane segments in the middle of the protein anchor NS4B to membranes. An amphipatic helix at the amino-terminus attaches to membranes as well. The carboxy-terminal domain (CTD) of NS4B is highly conserved in Hepaciviruses, though its function remains unknown. RESULTS: A cytosolic localization is predicted for the NS4B-CTD. However, using membrane floatation assays and immunofluorescence, we now show targeting of the NS4B-CTD to membranes. Furthermore, a profile-profile search, with an HCV NS4B-CTD multiple sequence alignment, indicates sequence similarity to the membrane binding domain of prokaryotic D-lactate dehydrogenase (d-LDH). The crystal structure of E. coli d-LDH suggests that the region similar to NS4B-CTD is located in the membrane binding domain (MBD) of d-LDH, implying analogy in membrane association. Targeting of d-LDH to membranes occurs via electrostatic interactions of positive residues on the outside of the protein with negative head groups of lipids. To verify that anchorage of d-LDH MBD and NS4B-CTD is analogous, NS4B-CTD mutants were designed to disrupt these electrostatic interactions. Membrane association was confirmed by swopping the membrane contacting helix of d-LDH with the corresponding domain of the 4B-CTD. Furthermore, the functionality of these residues was tested in the HCV replicon system. CONCLUSION: Together these data show that NS4B-CTD is associated to membranes, similar to the prokaryotic d-LDH MBD, and is important for replication.

Seroreactivity to epidermodysplasia verruciformis-related human papillomavirus types is associated with nonmelanoma skin cancer.

DNA from epidermodysplasia verruciformis-related human papillomavirus (EV-HPV) types is frequently found in nonmelanoma skin cancer (squamous and basal cell carcinoma). Epidemiological studies that investigate the relation between EV-HPV infection and nonmelanoma skin cancer are scarce. We designed a case-control study in which we looked for HPV infection in 540 cases with a history of skin cancer and 333 controls. By measuring seroreactivity to L1 virus-like particles of EV-HPV types 5, 8, 15, 20, 24, and 38 and the genital type HPV16 and by estimating the skin cancer relative risk among HPV seropositives, we analyzed whether EV-HPV serorecognition is associated with nonmelanoma skin cancer. Seroreactivity to five of the six EV-HPV types tested (HPV5, 8, 15, 20, and 24) was significantly increased in the squamous cell carcinoma cases. After adjusting for age and sex, the estimated squamous cell carcinoma relative risk was significantly increased in HPV8 and HPV38 seropositives [odds ratio (OR) = 14.7 (95% confidence interval (CI), 1.6-135) and OR = 3.0 (95% CI, 1.1-8.4), respectively]. The estimated relative risk for nodular and superficial multifocal basal cell carcinoma was also significantly increased in the HPV8 seropositives [OR = 9.2 (95% CI, 1.1-78.2) and OR = 17.3 (95% CI, 2.1-143), respectively] and in the HPV20 seropositives [OR = 3.2 (95% CI 1.3-7.9) and OR = 3.4 (95% CI 1.2-9.5), respectively]. The relative risk of developing malignant melanoma was not increased among HPV seropositives, and no associations were found for HPV16. Restricted analyses among the HPV seropositives only, to exclude distortion by interindividual differences in seroresponsiveness, underscored the significance of our findings. Restricted analyses among patients with skin cancer only, however, revealed that EV-HPV seropositivity was not significantly more present in patients with nonmelanoma skin cancer than in those with melanoma skin cancer. Taken together, our results indicate that EV-HPV serorecognition is nonspecifically associated with nonmelanoma skin cancer and suggest that EV-HPV-directed seroresponses are induced upon skin cancer formation, rather than upon infection.

Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage.

The genome organization and expression strategy of the newly identified severe acute respiratory syndrome coronavirus (SARS-CoV) were predicted using recently published genome sequences. Fourteen putative open reading frames were identified, 12 of which were predicted to be expressed from a nested set of eight subgenomic mRNAs. The synthesis of these mRNAs in SARS-CoV-infected cells was confirmed experimentally. The 4382- and 7073 amino acid residue SARS-CoV replicase polyproteins are predicted to be cleaved into 16 subunits by two viral proteinases (bringing the total number of SARS-CoV proteins to 28). A phylogenetic analysis of the replicase gene, using a distantly related torovirus as an outgroup, demonstrated that, despite a number of unique features, SARS-CoV is most closely related to group 2 coronaviruses. Distant homologs of cellular RNA processing enzymes were identified in group 2 coronaviruses, with four of them being conserved in SARS-CoV. These newly recognized viral enzymes place the mechanism of coronavirus RNA synthesis in a completely new perspective. Furthermore, together with previously described viral enzymes, they will be important targets for the design of antiviral strategies aimed at controlling the further spread of SARS-CoV.

Production of recombinant H1 parvovirus stocks devoid of replication-competent viruses.

Vector and helper plasmids for the production of recombinant H1 (rH1) parvovirus, an oncolytic virus and candidate vector for cancer gene therapy, were constructed with the aim of reducing the contamination of these preparations with replication-competent viruses (RCV). Split-helper plasmids were constructed by manipulating the splicing signals for the capsid proteins such that VP1 and VP2 were expressed from separate plasmids. H1 vectors with similarly mutated splice sites were packaged, using the split-helper plasmids, and the resulting recombinant H1 viruses were completely free of RCV because the generation of recombinants expressing both capsid proteins was prevented. Vector yields of rH1 produced with split-helper plasmids in combination with splice site-modified vectors were similar (in the range of 10(7) replication units/ml) to yields of rH1 produced with the standard vector/helper pair, in which case significant levels of RCV were generated (10(4)-10(5) plaque-forming units/ml). To assess the functionality of this approach in vivo, rH1 was produced that contained the human interleukin 2 (IL-2) transgene and that was devoid of RCV. This IL-2-carrying rH1 vector expressed IL-2 efficiently in human tumor cells (HeLa) in vitro and generated antitumor responses in nude mice xenografted with HeLa cells that had been infected ex vivo with this virus. These results should allow the large-scale production of recombinant oncotropic parvoviruses and their assessment for the gene therapy of cancer in a clinical setting.

Meta-analysis of mutations in the NS5A gene and hepatitis C virus resistance to interferon therapy: uniting discordant conclusions.

BACKGROUND: Hepatitis C virus genotype 1B responds poorly to treatment with interferon, in contrast to the more interferon-sensitive genotypes 2 and 3. Studies on combination therapy regimens with PEG-interferon and ribavirin report sustained response rates that generally do not exceed 50%, in contrast to sustained response rates of 80% for genotype 2 and 3. In Japan, a correlation was found between the number of mutations in an 'interferon sensitivity determining region' (ISDR) and outcome of interferon treatment in genotype 1B-infected patients. However, an ongoing controversy on the existence of an ISDR in non-Japanese isolates resulted, as non-Japanese studies failed to confirm this association. The present study approached this issue by carrying out a meta-analysis of ISDR sequences and response to interferon treatment. METHODS: Twenty-seven studies were included, reporting 1351 ISDR sequence data of genotype 1B-infected patients and their virological response to interferon treatment. Both summary statistics and individual patient data were used systematically to explore the association between ISDR mutations and response to interferon. RESULTS: The ISDR effect on response was universally present but appeared to be stronger in Japan, with a relative risk of 5.73 for mutant viruses as compared to 4.66 for non-Japanese isolates. High interferon dose, in Japan administered more frequently, was associated with an increase in response rate only among patients infected with mutant isolates. Interaction between dose and ISDR type was confirmed in a logistic regression model. After stratifying for dose, differences in response rate between Japanese and non-Japanese patients were no longer present. CONCLUSION: This study puts an end to a longstanding controversy by confirming the universal existence of an ISDR in genotype 1B-infected patients. Apparent discrepant findings from Japanese and non-Japanese studies can be explained by differences in dosing regimens and a dose-dependent differential effect of ISDR mutations on response to treatment.

The potentiating effect of ribavirin on interferon in the treatment of hepatitis C: lack of evidence for ribavirin-induced viral mutagenesis.

The recent finding that ribavirin has a mutagenic capacity in a poliovirus replicon model pushing the virus into error catastrophe, provides a possible explanation for the remarkable synergistic effect of ribavirin when combined with interferon in the treatment of chronic hepatitis C virus (HCV)-infected patients. However, ribavirin-induced hypermutation resulting in loss of vital genetic information and viral clearance, does not occur during treatment of HCV-infected patients, as can be inferred from the lack of viral inhibition when treating HCV-infected patients with ribavirin alone. We therefore hypothesized that ribavirin induces mutations in the C-terminal part of the viral NS5A gene, a region found to be correlated with interferon sensitivity. Ribavirin-induced mutations resulting in the appearance of viral variants more sensitive to interferon would explain the synergistic effect of ribavirin when combined with interferon. To test this hypothesis we retrospectively analysed sequences of the C-terminal half of the NS5A gene before and during treatment in six HCV genotype 1-infected patients who had been treated with combination therapy after initial failure to respond permanently to interferon alone. Our results show that during the early treatment phase mutation rate is not enhanced during combination therapy and that, at least in the major variant, shifts in the NS5A domain resulting in the occurrence of viral variants, which are more interferon-sensitive, do not occur.

Design and validation of consensus-degenerate hybrid oligonucleotide primers for broad and sensitive detection of corona- and toroviruses.

The ssRNA+ family Coronaviridae includes two subfamilies prototyped by coronaviruses and toroviruses that cause respiratory and enteric infections. To facilitate the identification of new distantly related members of the family Coronaviridae, we have developed a molecular assay with broad specificity. The consensus-degenerated hybrid oligonucleotide primer (CODEHOP) strategy was modified to design primers targeting the most conserved motifs in the RNA-dependent RNA polymerase locus. They were evaluated initially on RNA templates from virus-infected cells using a two-step RT-PCR protocol that was further advanced to a one-step assay. The sensitivity of the assay ranged from 10(2) to 10(6) and from 10(5) to 10(9) RNA copy numbers for individual corona-/torovirus templates when tested, respectively, with and without an excess of RNA from human cells. This primer set compared to that designed according to the original CODEHOP rules showed 10-10(3) folds greater sensitivity for 5 of the 6 evaluated corona-/torovirus templates. It detected 57% (32 of 56) of the respiratory specimens positive for 4 human coronaviruses, as well as stool specimens positive for a bovine torovirus. The high sensitivity and broad virus range of this assay makes it suitable for screening biological specimens in search for new viruses of the family Coronaviridae.

Mass spectrometry-based comparative sequencing to detect ganciclovir resistance in the UL97 gene of human cytomegalovirus.

BACKGROUND: Persistent infections with herpesviruses such as human cytomegalovirus (HCMV) frequently occur after solid organ or stem cell transplantation, and are due to either failure of the host to immunologically control the virus or emerging resistance of the virus to the antiviral drug(s) used. Antiviral therapy can be guided by viral drug susceptibility testing based on screening for known resistance-inducing mutations in the viral genome. Mass spectrometry-based comparative sequence analysis (MSCSA) might be advantageous for this purpose because of its suitability for semi-automation. OBJECTIVES: The applicability of MSCSA to detect sequence polymorphisms and drug resistance-inducing mutations in the HCMV genome was investigated. STUDY DESIGN: We analyzed the 3' part of the HCMV UL97 gene, which encodes the kinase that is activated by the commonly used anti-HCMV drug ganciclovir. Sequences obtained by MSCSA of material from HCMV-infected patients (43 samples) and the HCMV type strain were compared to conventional cycle sequencing results. RESULTS: In 94.1% of all samples the results obtained by MSCSA of the UL97 gene were identical to those from conventional cycle sequencing. The threshold to detect mutant sequences in a mixture with wild-type material was 20% using either technique. Furthermore, MSCSA was successfully applied to study the development of drug resistance in a patient who developed encephalitis due to ganciclovir-resistant HCMV. CONCLUSIONS: MSCSA was found to be equally accurate compared to conventional cycle sequencing in the analysis of UL97 of HCMV.

The coronavirus spike protein induces endoplasmic reticulum stress and upregulation of intracellular chemokine mRNA concentrations.

Murine hepatitis virus (MHV) and severe acute respiratory syndrome (SARS) coronavirus (CoV) are two of the best-studied representatives of the family Coronaviridae. During CoV infection, numerous cytokines and chemokines are induced in vitro and in vivo. Human interleukin 8 and its mouse functional counterpart, CXCL2, are early-expressed chemokines. Here we show that SARS-CoV and MHV induce endoplasmic reticulum (ER) stress and Cxcl2 mRNA transcription during infection in vitro. Expression of the viral spike protein significantly induced ER stress and Cxcl2 mRNA upregulation, while expression of the other structural genes did not. Additional experiments with UV-inactivated virus, cell-cell fusion-blocking antibodies, and an MHV mutant with a defect in spike protein maturation demonstrated that spike-host interactions in the ER are responsible for the induction of ER stress and subsequent Cxcl2 mRNA transcription. Despite significant increases in levels of Cxcl2 mRNA and functional nucleus-to-cytoplasm RNA transport, no CXCL2 protein was released into the medium from MHV-infected cells. Yet Sendai virus-infected cells showed substantial Cxcl2 mRNA induction and a simultaneous increase in levels of secreted CXCL2 protein. Our results demonstrate that expression of CoV spike proteins induces ER stress, which could subsequently trigger innate immune responses. However, at that point in infection, translation of host mRNA is already severely reduced in infected cells, preventing the synthesis of CXCL2 and ER stress proteins despite their increased mRNA concentrations.

Group 2 coronaviruses prevent immediate early interferon induction by protection of viral RNA from host cell recognition.

Many viruses encode antagonists to prevent interferon (IFN) induction. Infection of fibroblasts with the murine hepatitis coronavirus (MHV) and SARS-coronavirus (SARS-CoV) did not result in nuclear translocation of interferon-regulatory factor 3 (IRF3), a key transcription factor involved in IFN induction, and induction of IFN mRNA transcription. Furthermore, MHV and SARS-CoV infection could not prevent IFN induction by poly (I:C) or Sendai virus, suggesting that these CoVs do not inactivate IRF3-mediated transcription regulation, but apparently prevent detection of replicative RNA by cellular sensory molecules. Our data indicate that shielding of viral RNA to host cell sensors might be the main general mechanism for coronaviruses to prevent IFN induction.

Conservation of the pentanucleotide motif at the top of the yellow fever virus 17D 3' stem-loop structure is not required for replication.

The pentanucleotide (PN) sequence 5'-CACAG-3' at the top of the 3' stem-loop structure of the flavivirus genome is well conserved in the arthropod-borne viruses but is more variable in flaviviruses with no known vector. In this study, the sequence requirements of the PN motif for yellow fever virus 17D (YFV) replication were determined. In general, individual mutations at either the second, third or fourth positions were tolerated and resulted in replication-competent virus. Mutations at the fifth position were lethal. Base pairing of the nucleotide at the first position of the PN motif and a nucleotide four positions downstream of the PN (ninth position) was a major determinant for replication. Despite the fact that the majority of the PN mutants were able to replicate efficiently, they were outcompeted by parental YFV-17D virus following repeated passages in double-infected cell cultures. Surprisingly, some of the virus mutants at the first and/or the ninth position that maintained the possibility of forming a base pair were found to have a similar fitness to YFV-17D under these conditions. Overall, these experiments suggest that YFV is less dependent on sequence conservation of the PN motif for replication in animal cells than West Nile virus. However, in animal cell culture, YFV has a preference for the wt CACAG PN sequence. The molecular mechanisms behind this preference remain to be elucidated.

Vaccine-induced early control of hepatitis C virus infection in chimpanzees fails to impact on hepatic PD-1 and chronicity.

UNLABELLED: Broad T cell and B cell responses to multiple HCV antigens are observed early in individuals who control or clear HCV infection. The prevailing hypothesis has been that similar immune responses induced by prophylactic immunization would reduce acute virus replication and protect exposed individuals from chronic infection. Here, we demonstrate that immunization of naïve chimpanzees with a multicomponent HCV vaccine induced robust HCV-specific immune responses, and that all vaccinees exposed to heterologous chimpanzee-adapted HCV 1b J4 significantly reduced viral RNA in serum by 84%, and in liver by 99% as compared to controls (P=0.024 and 0.028, respectively). However, despite control of HCV in plasma and liver in the acute period, in the chronic phase, 3 of 4 vaccinated animals developed persistent infection. Analysis of expression levels of proinflammatory cytokines in serial hepatic biopsies failed to reveal an association with vaccine outcome. However, expression of IDO, CTLA-4 [corrected] and PD-1 levels in liver correlated with clearance or chronicity. CONCLUSION: Despite early control of virus load, a virus-associated tolerogenic-like state can develop in certain individuals independent of vaccination history.

Transcriptional profiling of acute cytopathic murine hepatitis virus infection in fibroblast-like cells.

Understanding the orchestrated genome-wide cellular responses is critical for comprehending the early events of coronavirus infection. Microarray analysis was applied to assess changes in cellular expression profiles during different stages of two independent, highly controlled murine hepatitis virus (MHV) infections in vitro. Fibroblast-like L cells were infected at high multiplicity in order to study the direct effects of a synchronized lytic coronavirus infection. Total RNA was harvested from MHV- or mock-infected L cells at 3, 5 and 6 h post-infection and hybridized to Affymetrix microarrays representing approximately 12,500 murine genes and expressed sequences. The expression data were compared to their respective mock-infected controls. Quantitative RT-PCR of selected transcripts was used to validate the differential expression of transcripts and inter-experiment reproducibility of microarray analysis. It was concluded that MHV-A59 infection in fibroblast-like cells triggers very few transcriptional cellular responses in the first 3 h of infection. Later, after having established a productive infection, a chemokine response is induced together with other cellular changes associated with RNA and protein metabolism, cell cycle and apoptosis. Interferon responses are not triggered during infection, although the L cells can be readily stimulated to produce interferon by dsRNA, a known potent inducer of interferon. Possibly, the interferon response is actively counteracted by a virus-encoded antagonist as has been described previously for other RNA viruses.

Nidovirus transcription: how to make sense...?

Many positive-stranded RNA viruses use subgenomic mRNAs to express part of their genetic information. To produce structural and accessory proteins, members of the order Nidovirales (corona-, toro-, arteri- and roniviruses) generate a 3' co-terminal nested set of at least three and often seven to nine mRNAs. Coronavirus and arterivirus subgenomic transcripts are not only 3' co-terminal but also contain a common 5' leader sequence, which is derived from the genomic 5' end. Their synthesis involves a process of discontinuous RNA synthesis that resembles similarity-assisted RNA recombination. Most models proposed over the past 25 years assume co-transcriptional fusion of subgenomic RNA leader and body sequences, but there has been controversy over the question of whether this occurs during plus- or minus-strand synthesis. In the latter model, which has now gained considerable support, subgenomic mRNA synthesis takes place from a complementary set of subgenome-size minus-strand RNAs, produced by discontinuous minus-strand synthesis. Sense-antisense base-pairing interactions between short conserved sequences play a key regulatory role in this process. In view of the presumed common ancestry of nidoviruses, the recent finding that ronivirus and torovirus mRNAs do not contain a common 5' leader sequence is surprising. Apparently, major mechanistic differences must exist between nidoviruses, which raises questions about the functions of the common leader sequence and nidovirus transcriptase proteins and the evolution of nidovirus transcription. In this review, nidovirus transcription mechanisms are compared, the experimental systems used are critically assessed and, in particular, the impact of recently developed reverse genetic systems is discussed.

Repair and polyadenylation of a naturally occurring hepatitis C virus 3' nontranslated region-shorter variant in selectable replicon cell lines.

The 3' nontranslated region (NTR) of the hepatitis C virus (HCV) genome is highly conserved and contains specific cis-acting RNA motifs that are essential in directing the viral replication machinery to initiate at the correct 3' end of the viral genome. Since the ends of viral genomes may be damaged by cellular RNases, preventing the initiation of viral RNA replication, stable RNA hairpin structures in the 3' NTR may also be essential in host defense against exoribonucleases. During 3'-terminal sequence analysis of serum samples of a patient with chronic hepatitis related to an HCV1b infection, a number of clones were obtained that were several nucleotides shorter at the extreme 3' end of the genome. These shorter 3' ends were engineered in selectable HCV replicons in order to enable the study of RNA replication in cell culture. When in vitro-transcribed subgenomic RNAs, containing shorter 3' ends, were introduced into Huh-7 cells, a few selectable colonies were obtained, and the 3' terminus of these subgenomic RNAs was sequenced. Interestingly, most genomes recovered from these colonies had regained the wild-type 3' ends, showing that HCV, like several other positive-stranded RNA viruses, has developed a strategy to repair deleted 3' end nucleotides. Furthermore, we found several genomes in these replicon colonies that contained a poly(A) tail and a short linker sequence preceding the poly(A) tail. After recloning and subsequent passage in Huh-7 cells, these poly(A) tails persisted and varied in length. In addition, the connecting linker became highly diverse in sequence and length, suggesting that these tails are actively replicated. The possible terminal repair mechanisms, including roles for the poly(A) tail addition, are discussed.