Increased pathogenicity and aerosol transmission for one SARS-CoV-2 B.1.617.2 Delta variant over the wild-type strain in hamsters

During the two-year pandemic of coronavirus disease 2019 (COVID-19), its causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been evolving. SARS-CoV-2 Delta, a variant of concern, has become the dominant circulating strain worldwide within just a few months. Here, we performed a comprehensive analysis of a new B.1.617.2 Delta strain (Delta630) compared with the early WIV04 strain (WIV04) in vitro and in vivo, in terms of replication, infectivity, pathogenicity, and transmission in hamsters. When inoculated intranasally, Delta630 led to more pronounced weight loss and more severe disease in hamsters. Moreover, 40% mortality occurred about one week after infection with 10⁴ PFU of Delta630, whereas no deaths occurred even after infection with 10⁵ PFU of WIV04 or other strains belonging to the Delta variant. Moreover, Delta630 outgrew over WIV04 in the competitive aerosol transmission experiment. Taken together, the Delta630 strain showed increased replication ability, pathogenicity, and transmissibility over WIV04 in hamsters. To our knowledge, this is the first SARS-CoV-2 strain that causes death in a hamster model, which could be an asset for the efficacy evaluation of vaccines and antivirals against infections of SARS-CoV-2 Delta strains. The underlying molecular mechanisms of increased virulence and transmission await further analysis.     

Characteristics of replication and pathogenicity of SARS-CoV-2 Alpha and Delta isolates

The continuously arising of SARS-CoV-2 variants has been posting a great threat to public health safety globally, from B.1.17 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.617.2 (Delta) to B.1.1.529 (Omicron). The emerging or reemerging of the SARS-CoV-2 variants of concern is calling for the constant monitoring of their epidemics, pathogenicity and immune escape. In this study, we aimed to characterize replication and pathogenicity of the Alpha and Delta variant strains isolated from patients infected in Laos. The amino acid mutations within the spike fragment of the isolates were determined via sequencing. The more efficient replication of the Alpha and Delta isolates was documented than the prototyped SARS-CoV-2 in Calu-3 and Caco-2 cells, while such features were not observed in Huh-7, Vero E6 and HPA-3 cells. We utilized both animal models of human ACE2 (hACE2) transgenic mice and hamsters to evaluate the pathogenesis of the isolates. The Alpha and Delta can replicate well in multiple organs and cause moderate to severe lung pathology in these animals. In conclusion, the spike protein of the isolated Alpha and Delta variant strains was characterized, and the replication and pathogenicity of the strains in the cells and animal models were also evaluated.     

Nasal delivery of broadly neutralizing antibodies protects mice from lethal challenge with SARS-CoV-2 delta and omicron variants

Multiple new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have constantly emerged, as the delta and omicron variants, which have developed resistance to currently gained neutralizing antibodies. This highlights a critical need to discover new therapeutic agents to overcome the variants mutations. Despite the availability of vaccines against coronavirus disease 2019 (COVID-19), the use of broadly neutralizing antibodies has been considered as an alternative way for the prevention or treatment of SARS-CoV-2 variants infection. Here, we show that the nasal delivery of two previously characterized broadly neutralizing antibodies (F61 and H121) protected K18-hACE2 mice against lethal challenge with SARS-CoV-2 variants. The broadly protective efficacy of the F61 or F61/F121 cocktail antibodies was evaluated by lethal challenge with the wild strain (WIV04) and multiple variants, including beta (B.1.351), delta (B.1.617.2), and omicron (B.1.1.529) at 200 or 1000 TCID50, and the minimum antibody administration doses (5-1.25 mg/kg body weight) were also evaluated with delta and omicron challenge. Fully prophylactic protections were found in all challenged groups with both F61 and F61/H121 combination at the administration dose of 20 mg/kg body weight, and corresponding mice lung viral RNA showed negative, with almost all alveolar septa and cavities remaining normal. Furthermore, low-dose antibody treatment induced significant prophylactic protection against lethal challenge with delta and omicron variants, whereas the F61/H121 combination showed excellent results against omicron infection. Our findings indicated the potential use of broadly neutralizing monoclonal antibodies as prophylactic and therapeutic agent for protection of current emerged SARS-CoV-2 variants infection.     

Neutralization of five SARS-CoV-2 variants of concern by convalescent and BBIBP-CorV vaccinee serum

The prevalence of SARS-CoV-2 variants of concern (VOCs) is still escalating throughout the world. However, the level of neutralization of the inactivated viral vaccine recipients’ sera and convalescent sera against all VOCs, including B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.617.2 (Delta), and B.1.1.529 (Omicron) remains to be lack of comparative analysis. Therefore, we constructed pseudoviruses of five VOCs using a lentiviral-based system and analyzed their viral infectivity and neutralization resistance to convalescent and BBIBP-CorV vaccinee serum at different times. Our results show that, compared with the wild-type strain (WT), five VOC pseudoviruses showed higher infection, of which B.1.617.2 and B.1.1.529 variant pseudoviruses exhibited higher infection rates than wild-type or other VOC strains, respectively. Sera from 10 vaccinated individuals at the 1, 3 and 5-month post second dose or from 10 convalescent at 14 and 200 days after discharge retained neutralizing activity against all strains but exhibited decreased neutralization activity significantly against the five VOC variant pseudoviruses over time compared to WT. Notably, 100% (30/30) of the vaccinee serum samples showed more than a 2.5-fold reduction in neutralizing activity against B.1.1.529, and 90% (18/20) of the convalescent serum samples showed more than 2.5-fold reduction in neutralization against B.1.1.529. These findings demonstrate the reduced protection against the VOCs in vaccinated and convalescent individuals over time, indicating that it is necessary to have a booster shot and develop new vaccines capable of eliciting broad neutralization antibodies.     

Host microRNAs exhibit differential propensity to interact with SARS-CoV-2 and variants of concern

A significant number of SARS-CoV-2-infected individuals naturally overcome viral infection, suggesting the existence of a potent endogenous antiviral mechanism. As an innate defense mechanism, microRNA (miRNA) pathways in mammals have evolved to restrict viruses, besides regulating endogenous mRNAs. In this study, we systematically examined the complete repertoire of human miRNAs for potential binding sites on SARS-CoV-2 Wuhan-Hu-1, Beta, Delta, and Omicron. Human miRNA and viral genome interaction were analyzed using RNAhybrid 2.2 with stringent parameters to identify highly bonafide miRNA targets. Using publicly available data, we filtered for miRNAs expressed in lung epithelial cells/tissue and oral keratinocytes, concentrating on the miRNAs that target SARS-CoV-2 S protein mRNAs. Our results show a significant loss of human miRNA and SARS-CoV-2 interactions in Omicron (130 miRNAs) compared to Wuhan-Hu-1 (271 miRNAs), Beta (279 miRNAs), and Delta (275 miRNAs). In particular, hsa-miR-3150b-3p and hsa-miR-4784 show binding affinity for S protein of Wuhan strain but not Beta, Delta, and Omicron. Loss of miRNA binding sites on N protein was also observed for Omicron. Through Ingenuity Pathway Analysis (IPA), we examined the experimentally validated and highly predicted functional role of these miRNAs. We found that hsa-miR-3150b-3p and hsa-miR-4784 have several experimentally validated or highly predicted target genes in the Toll-like receptor, IL-17, Th1, Th2, interferon, and coronavirus pathogenesis pathways. Focusing on the coronavirus pathogenesis pathway, we found that hsa-miR-3150b-3p and hsa-miR-4784 are highly predicted to target MAPK13. Exploring miRNAs to manipulate viral genome/gene expression can provide a promising strategy with successful outcomes by targeting specific VOCs.     

Incorporation of dengue virus replicon into virus-like particles by a cell line stably expressing precursor membrane and envelope proteins of dengue virus type 2

While virus-like particles (VLPs) containing subgenomic replicons, which can transduce replicons into target cells efficiently for studying viral replication and vectors of gene therapy and vaccine, have been established for several flaviviruses, none has been reported for the four serotypes of dengue virus, the causal agent of the most important arboviral diseases in this century. In this study, we successfully established a cell line stably expressing the precursor membrane/envelope (PrM/E) proteins of dengue virus type 2 (DENV2), which can package a DENV2 replicon with deletion of PrM/E genes and produce single-round infectious VLPs. Moreover, it can package a similar replicon of different serotype, dengue virus type 4, and produce infectious chimeric VLPs. To our knowledge, this study reports for the first time replicon-containing VLPs of dengue virus. Moreover, this convenient system has potential as a valuable tool to study encapsidation of dengue virus and to develop novel chimeric VLPs containing dengue virus replicon as vaccine in the future.     

Clinical characteristics of 310 SARS-CoV-2 Omicron variant patients and comparison with Delta and Beta variant patients in China

Although the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant has spread, data on the clinical characteristics of infected patients are limited. In this study, the demographic, clinical characteristics, and laboratory data of 310 SARS-CoV-2 Omicron variant patients treated at Haihe Hospital of Tianjin were collected and analyzed. Information on these patients was compared to 96 patients with the Delta variant of concern (VOC) and 326 patients with the Beta VOC during the previous coronavirus disease 2019 (COVID-19) outbreak in Harbin. Of the 310 patients infected with the Omicron variant, the median age was 35 years. Most patients were clinically classified as mild (57.74%), and the most common symptoms were cough (48.71%), fever (39.35%), and sore throat (38.26%). The results for different vaccination groups in the Omicron group showed that the median of “SARS-CoV-2 specific IgG” after 2 or 3 doses of vaccination was higher than the unvaccinated group (all Ps < 0.05). Older age was associated with a higher proportion of moderate cases and lower asymptomatic and mild cases based on clinical classifications. Compared to the Delta and Beta groups, the median age of the Omicron group was younger. The total number of asymptomatic patients and mild patients in the Omicron virus group was higher than the Delta and Beta groups (60.97% vs. 54.17% vs. 47.55%). This study presented the clinical characteristics of the first group of patients infected with the Omicron variant in Tianjin, China, and compared their clinical features with patients infected by the Delta and Beta variants, which would increase our understanding of the characteristics of SARS-CoV-2 Omicron variant.     

Replicon Clusters Are Stable Units of Chromosome Structure: Evidence That Nuclear Organization Contributes to the Efficient Activation and Propagation of S Phase in Human Cells

In proliferating cells, DNA synthesis must be performed with extreme precision. We show that groups of replicons, labeled together as replicon clusters, form stable units of chromosome structure. HeLa cells were labeled with 5-bromodeoxyuridine (BrdU) at different times of S phase. At the onset of S phase, clusters of replicons were activated in each of ~750 replication sites. The majority of these replication “foci” were shown to be individual replicon clusters that remained together, as stable cohorts, throughout the following 15 cell cycles. In individual cells, the same replication foci were labeled with BrdU and 5-iododeoxyuridine at the beginning of different cell cycles. In DNA fibers, 95% of replicons in replicon clusters that were labeled at the beginning of one S phase were also labeled at the beginning of the next. This shows that a subset of origins are activated both reliably and efficiently in different cycles.

The majority of replication forks activated at the onset of S phase terminated 45–60 min later. During this interval, secondary replicon clusters became active. However, while the activation of early replicons is synchronized at the onset of S phase, different secondary clusters were activated at different times. Nevertheless, replication foci pulse labeled during any short interval of S phase were stable for many cell cycles. We propose that the coordinated replication of related groups of replicons, that form stable replicon clusters, contributes to the efficient activation and propagation of S phase in mammalian cells.

     

Evaluation of the relative virulence of novel SARS-CoV-2 variants: a retrospective cohort study in Ontario,Canada

Background:Between February and June 2021, the initial wild-type strains of SARS-CoV-2 were supplanted in Ontario, Canada, by new variants of concern (VOCs), first those with the N501Y mutation (i.e., Alpha/B1.1.17, Beta/B.1.351 and Gamma/P.1 variants) and then the Delta/B.1.617 variant. The increased transmissibility of these VOCs has been documented, but knowledge about their virulence is limited. We used Ontario’s COVID-19 case data to evaluate the virulence of these VOCs compared with non-VOC SARS-CoV-2 strains, as measured by risk of hospitalization, intensive care unit (ICU) admission and death.Methods:We created a retrospective cohort of people in Ontario who tested positive for SARS-CoV-2 and were screened for VOCs, with dates of test report between Feb. 7 and June 27, 2021. We constructed mixed-effect logistic regression models with hospitalization, ICU admission and death as outcome variables. We adjusted models for age, sex, time, vaccination status, comorbidities and pregnancy status. We included health units as random intercepts.Results:Our cohort included 212 326 people. Compared with non-VOC SARS-CoV-2 strains, the adjusted elevation in risk associated with N501Y-positive variants was 52% (95% confidence interval [CI] 42%–63%) for hospitalization, 89% (95% CI 67%–117%) for ICU admission and 51% (95% CI 30%–78%) for death. Increased risk with the Delta variant was more pronounced at 108% (95% CI 78%–140%) for hospitalization, 235% (95% CI 160%–331%) for ICU admission and 133% (95% CI 54%–231%) for death.Interpretation:The increasing virulence of SARS-CoV-2 VOCs will lead to a considerably larger, and more deadly, pandemic than would have occurred in the absence of the emergence of VOCs.

Novel SARS-CoV-2 variants of concern (VOCs), including viral lineages carrying the N501Y (Alpha/B.1.1.7) or both the N501Y and E484K mutations (Beta/B.1.351 and Gamma/P.1), were first identified in Ontario, Canada, in December 2020.¹ Although initially uncommon in Ontario, these VOCs outcompeted earlier SARS-CoV-2 lineages and, as of late April 2021, were responsible for almost all new infections in Ontario, with Alpha the most prevalent lineage.¹ In April 2021, the B.1.617.2 variant, now known as Delta under the revised nomenclature from the World Health Organization, emerged in the province, outcompeted earlier VOCs and, by July 2021, represented most infections in the province.^2,3This serial replacement by emerging variants reflects progressively higher effective reproduction numbers that allow novel variants to outcompete previously dominant strains in the face of identical measures to control spread of infection.^4–6 However, VOCs are also concerning because emerging evidence points to increased virulence, with increased risk of hospitalization, intensive care unit (ICU) admission and death, after adjustment for age and other predictive factors among patients with VOC infections.^7–10 Although the increased virulence of strains with the N501Y mutation relative to strains that lack this mutation has been described,^7–9 only limited information is available on the virulence of infection with the Delta variant, relative to earlier N501Y-positive VOCs (i.e., Alpha, Beta and Gamma).^10–12 Our objectives were to evaluate the virulence of N501Y-positive variants relative to earlier SARS-CoV-2 lineages and to evaluate the virulence of the Delta variant of SARS-CoV-2 relative to N501Y-positive VOCs using Ontario’s COVID-19 case data.     

Selection and characterization of replicon variants dually resistant to thumb- and palm-binding nonnucleoside polymerase inhibitors of the hepatitis C virus

Multiple nonnucleoside inhibitor binding sites have been identified within the hepatitis C virus (HCV) polymerase, including in the palm and thumb domains. After a single treatment with a thumb site inhibitor (thiophene-2-carboxylic acid NNI-1), resistant HCV replicon variants emerged that contained mutations at residues Leu419, Met423, and Ile482 in the polymerase thumb domain. Binding studies using wild-type (WT) and mutant enzymes and structure-based modeling showed that the mechanism of resistance is through the reduced binding of the inhibitor to the mutant enzymes. Combined treatment with a thumb- and a palm-binding polymerase inhibitor had a dramatic impact on the number of replicon colonies able to replicate in the presence of both inhibitors. A more exact characterization through molecular cloning showed that 97.7% of replicons contained amino acid substitutions that conferred resistance to either of the inhibitors. Of those, 65% contained simultaneously multiple amino acid substitutions that conferred resistance to both inhibitors. Double-mutant replicons Met414Leu and Met423Thr were predominantly selected, which showed reduced replication capacity compared to the WT replicon. These findings demonstrate the selection of replicon variants dually resistant to two NS5B polymerase inhibitors binding to different sites of the enzyme. Additionally, these findings provide initial insights into the in vitro mutational threshold of the HCV NS5B polymerase and the potential impact of viral fitness on the selection of multiple-resistant mutants.     

Effect of alpha interferon on the hepatitis C virus replicon

Chronic hepatitis C virus (HCV) infections can be cured only in a fraction of patients treated with alpha interferon (IFN-alpha) and ribavirin combination therapy. The mechanism of the IFN-alpha response against HCV is not understood, but evidence for a role for viral nonstructural protein 5A (NS5A) in IFN resistance has been provided. To elucidate the mechanism by which NS5A and possibly other viral proteins inhibit the cellular antiviral program, we have constructed a subgenomic replicon from a known infectious HCV clone and demonstrated that it has an approximately 1,000-fold-higher transduction efficiency than previously used subgenomes. We found that IFN-alpha reduced replication of HCV subgenomic replicons approximately 10-fold. The estimated half-life of viral RNA in the presence of the cytokine was about 12 h. HCV replication was sensitive to IFN-alpha independently of whether the replicon expressed an NS5A protein associated with sensitivity or resistance to the cytokine. Furthermore, our results indicated that HCV replicons can persist in Huh7 cells in the presence of high concentrations of IFN-alpha. Finally, under our conditions, selection for IFN-alpha-resistant variants did not occur.     

Construction of a chimeric hepatitis C virus replicon based on a strain isolated from a chronic hepatitis C patient

Subgenomic replicons of hepatitis C virus (HCV) have been widely used for studying HCV replication. Here, we report a new subgenomic replicon based on a strain isolated from a chronically infected patient. The coding sequence of HCV was recovered from a Chinese chronic hepatitis C patient displaying high serum HCV copy numbers. A consensus sequence designated as CCH strain was constructed based on the sequences of five clones and this was classified by sequence alignment as belonging to genotype 2a. The subgenomic replicon of CCH was replication-deficient in cell culture, due to dysfunctions in NS3 and NS5B. Various JFH1/CCH chimeric replicons were constructed, and specific mutations were introduced. The introduction of mutations could partially restore the replication of chimeric replicons. A replication-competent chimeric construct was finally obtained by the introduction of NS3 from JFH1 into the backbone of the CCH strain.     

A Drosophila Toolkit for the Visualization and Quantification of Viral Replication Launched from Transgenic Genomes

Arthropod RNA viruses pose a serious threat to human health, yet many aspects of their replication cycle remain incompletely understood. Here we describe a versatile Drosophila toolkit of transgenic, self-replicating genomes (‘replicons’) from Sindbis virus that allow rapid visualization and quantification of viral replication in vivo. We generated replicons expressing Luciferase for the quantification of viral replication, serving as useful new tools for large-scale genetic screens for identifying cellular pathways that influence viral replication. We also present a new binary system in which replication-deficient viral genomes can be activated ‘in trans’, through co-expression of an intact replicon contributing an RNA-dependent RNA polymerase. The utility of this toolkit for studying virus biology is demonstrated by the observation of stochastic exclusion between replicons expressing different fluorescent proteins, when co-expressed under control of the same cellular promoter. This process is analogous to ‘superinfection exclusion’ between virus particles in cell culture, a process that is incompletely understood. We show that viral polymerases strongly prefer to replicate the genome that encoded them, and that almost invariably only a single virus genome is stochastically chosen for replication in each cell. Our in vivo system now makes this process amenable to detailed genetic dissection. Thus, this toolkit allows the cell-type specific, quantitative study of viral replication in a genetic model organism, opening new avenues for molecular, genetic and pharmacological dissection of virus biology and tool development.     

Novel cell culture-adapted genotype 2a hepatitis C virus infectious clone

Although the recently developed infectious hepatitis C virus system that uses the JFH-1 clone enables the study of whole HCV viral life cycles, limited particular HCV strains have been available with the system. In this study, we isolated another genotype 2a HCV cDNA, the JFH-2 strain, from a patient with fulminant hepatitis. JFH-2 subgenomic replicons were constructed. HuH-7 cells transfected with in vitro transcribed replicon RNAs were cultured with G418, and selected colonies were isolated and expanded. From sequencing analysis of the replicon genome, several mutations were found. Some of the mutations enhanced JFH-2 replication; the 2217AS mutation in the NS5A interferon sensitivity-determining region exhibited the strongest adaptive effect. Interestingly, a full-length chimeric or wild-type JFH-2 genome with the adaptive mutation could replicate in Huh-7.5.1 cells and produce infectious virus after extensive passages of the virus genome-replicating cells. Virus infection efficiency was sufficient for autonomous virus propagation in cultured cells. Additional mutations were identified in the infectious virus genome. Interestingly, full-length viral RNA synthesized from the cDNA clone with these adaptive mutations was infectious for cultured cells. This approach may be applicable for the establishment of new infectious HCV clones.     

昆津病毒复制子——一种新型病毒载体

病毒复制子 (Replicon) 是指来源于病毒基因组的能够自主复制的RNA分子,保留了病毒非结构蛋白基因,而结构蛋白基因缺失或由外源基因替代。昆津病毒 (Kunjun virus) 为黄病毒科黄病毒属成员,其复制子具有表达效率高、细胞毒性低、遗传稳定等特点,在病毒基因组复制调控机制、外源蛋白表达、新型疫苗和基因治疗等领域得到了广泛应用。以下就昆津病毒复制子系统的构建、特性及应用方面的研究进展作一综述。     

Cyclophilin A is an essential cofactor for hepatitis C virus infection and the principal mediator of cyclosporine resistance in vitro

Cyclosporine (CsA) and its derivatives potently suppress hepatitis C virus (HCV) replication. Recently, CsA-resistant HCV replicons have been identified in vitro. We examined the dependence of the wild-type and CsA-resistant replicons on various cyclophilins for replication. A strong correlation between CsA resistance and reduced dependency on cyclophilin A (CyPA) for replication was identified. Silencing of CyPB or CyPC expression had no significant effect on replication, whereas various forms of small interfering RNA (siRNA) directed at CyPA inhibited HCV replication of wild-type but not CsA-resistant replicons. The efficiency of a particular siRNA in suppressing CyPA expression was correlated with its potency in inhibiting HCV replication, and expression of an siRNA-resistant CyPA cDNA rescued replication. In addition, an anti-CyPA antibody blocked replication of the wild-type but not the resistant replicon in an in vitro replication assay. Depletion of CyPA alone in the CsA-resistant replicon cells eliminated CsA resistance, indicating that CyPA is the chief mediator of the observed CsA resistance. The dependency on CyPA for replication was observed for both genotype (GT) 1a and 1b replicons as well as a GT 2a infectious virus. An interaction between CyPA and HCV RNA as well as the viral polymerase that is sensitive to CsA treatment in wild-type but not in resistant replicons was detected. These findings reveal the molecular mechanism of CsA resistance and identify CyPA as a critical cellular cofactor for HCV replication and infection.     

Two independent replicons can support replication of the anthrax toxin-encoding plasmid pXO1 of Bacillus anthracis

The large pXO1 plasmid (181.6kb) of Bacillus anthracis encodes the anthrax toxin proteins. Previous studies have shown that two separate regions of pXO1 can support replication of pXO1 miniplasmids when introduced into plasmid-less strains of this organism. No information is currently available on the ability of the above two replicons, termed RepX and ORFs 14/16 replicons, to support replication of the full-length pXO1 plasmid. We generated mutants of the full-length pXO1 plasmid in which either the RepX or the ORFs 14/16 replicon was inactivated by TargeTron insertional mutagenesis. Plasmid pXO1 derivatives containing only the RepX or the ORFs 14/16 replicon were able to replicate when introduced into a plasmid-less B. anthracis strain. Plasmid copy number analysis showed that the ORFs 14/16 replicon is more efficient than the RepX replicon. Our studies demonstrate that both the RepX and ORFs 14/16 replicons can independently support the replication of the full-length pXO1 plasmid.     

Transcription strategy in a Closterovirus: a novel 5'-proximal controller element of Citrus Tristeza Virus produces 5'- and 3'-terminal subgenomic RNAs and differs from 3' open reading frame controller elements

Citrus tristeza virus (CTV) produces more than thirty 3'- or 5'-terminal subgenomic RNAs (sgRNAs) that accumulate to various extents during replication in protoplasts and plants. Among the most unusual species are two abundant populations of small 5'-terminal sgRNAs of approximately 800 nucleotides (nt) termed low-molecular-weight tristeza (LMT1 and LMT2) RNAs. Remarkably, CTV replicons with all 10 3' genes deleted produce only the larger LMT1 RNAs. These 5'-terminal positive-sense sgRNAs do not have corresponding negative strands and were hypothesized to be produced by premature termination during plus-strand genomic RNA synthesis. We characterized a cis-acting element that controls the production of the LMT1 RNAs. Since manipulation of this cis-acting element in its native position (the L-ProI region of replicase) was not possible because the mutations negatively affect replication, a region (5'TR) surrounding the putative termination sites (nt approximately 550 to 1000) was duplicated in the 3' end of a CTV replicon to allow characterization. The duplicated sequence continued to produce a 5'-terminal plus-strand sgRNA, here much larger ( approximately 11 kb), apparently by termination. Surprisingly, a new 3'-terminal sgRNA was observed from the duplicated 5'TR. A large 3'-terminal sgRNA resulting from the putative promoter activity of the native 5'TR was not observed, possibly because of the down-regulation of a promoter approximately 19 kb from the 3' terminus. However, we were able to observe a sgRNA produced from the native 5'TR of a small defective RNA, which placed the native 5'TR closer to the 3' terminus, demonstrating sgRNA promoter activity of the native 5'TR. Deletion mutagenesis mapped the promoter and the terminator activities of the 5'TR (in the 3' position in the CTV replicon) to a 57-nt region, which was folded by the MFOLD computer program into two stem-loops. Mutations in the putative stem-loop structures equally reduced or prevented production of both the 3'- and 5'-terminal sgRNAs. These mutations, when introduced in frame in the native 5'TR, similarly abolished the synthesis of the LMT1 RNAs and presumably the large 3'-terminal sgRNA while having no impact on replication, demonstrating that neither 5'- nor 3'-terminal sgRNA is necessary for replication of the replicon or full-length CTV in protoplasts. Differences between the 5'TR, which produced two plus-strand sgRNAs, and the cis-acting elements controlling the 3' open reading frames, which produced additional minus-strand sgRNAs corresponding to the 3'-terminal mRNAs, suggest that the different sgRNA controller elements had different origins in the modular evolution of closteroviruses.     

Molecular Characterization of Plasmid pBM300 from Bacillus megaterium QM B1551

Strain QM B1551 of Bacillus megaterium contains seven compatible plasmids: two small rolling circle plasmids and five theta-replicating plasmids with cross-hybridizing replicons. To expand our understanding of these plasmids, the replicon region (6.7 kb) from pBM300 was cloned, sequenced, and functionally characterized. Sequence analysis showed that the replication protein (RepM300) was highly homologous to two other plasmid Rep proteins of the same strain but to no other known proteins. Furthermore, the location of the replication origin was within the RepM300 coding region, and the origin contained three 12-base direct repeats. Deletion analysis of the replicon confirmed the role of the Rep protein and showed that open reading frame 2 (ORF2) was required for stability. However, the protein encoded by ORF2 is entirely different from the replicon stability proteins encoded by the other two replicons. The entire plasmid was isolated from the plasmid array by integrating a spectinomycin resistance gene and transforming a plasmidless strain, PV361. Complete sequencing showed that pBM300 was 26,300 bp long, had a G+C content of 35.2%, and contained 20 ORFs, two of which encoded proteins that had no similarity to other proteins in the database. The proteins encoded by the plasmid ORFs had similarity to proteins for mobilization and transfer, an integrase, a rifampin resistance protein, a cell wall hydrolase, glutathione synthase, and a biotin carboxylase. The similarities were to several gram-positive genera and a few gram-negative genera and archaea. oriT and ssoT-like regions were detected near two mob genes. These results suggest that pBM300 is a mobilizable hybrid plasmid that confers increased metabolic and germination ability on its host. Its replicon also helps define a new plasmid family.