Rubella Virus DI RNAs and Replicons: Requirement for Nonstructural Proteins Acting in Cis for Amplification by Helper Virus

Tzeng, Wen-Pin; Chen, Min-hsin; Derdeyn, Cynthia A.; Frey, Teryl K.

doi:10.1006/viro.2001.1088

Cited by 38 publications

(69 citation statements)

References 27 publications

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“…Finally, as discussed previously (32), the development of RUB replicons makes possible the generation of suicide vectors which cannot spread from cell to cell for delivery and expression of foreign genes. Such a system will require an efficient packaging systems for the replicons.…”

Section: Discussionmentioning

confidence: 96%

“…4). This replicon offered the advantage over replicons which did not contain an antibiotic resistance gene (32) of an easy assay for replication: determining the number of cells surviving antibiotic selection (relative survival). Additionally, cotransfection with helper viruses and amplification by passaging was not necessary (16), and replicon-specific macromolecular synthesis could be studied in the cells that survived selection and were thus enriched for the presence of the replicon.…”

Section: Discussionmentioning

confidence: 99%

“…For this reason, DI cDNA clones are commonly used since they are smaller than infectious clones and deletions can be made without regard to protein-encoding sequences (16). RUB replicons in which reporter genes replace the SP-ORF, thus mimicking RUB DI RNAs, have been developed (32). In this study, we created a RUB replicon in which an antibiotic resistance gene replaced most of the SP-ORF and which is thus capable of generating stable, replicon-containing cell lines following antibiotic selection.…”

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confidence: 99%

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Analysis of the 3′ cis -Acting Elements of Rubella Virus by Using Replicons Expressing a Puromycin Resistance Gene

Chen

Frolov

Icenogle

et al. 2004

J Virol

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A rubella virus (RUB) replicon, RUBrep/PAC, was constructed and used to map the 3 cis-acting elements (3 CSE) of the RUB genome required for RUB replication. The RUBrep/PAC replicon had the structural protein open reading frame partially replaced by a puromycin acetyltransferase (PAC) gene. Cells transfected with RUBrep/PAC transcripts expressed the PAC gene from the subgenomic RNA, were rendered resistant to puromycin, and thus survived selection with this drug. The relative survival following puromycin selection of cells transfected with transcripts from RUBrep/PAC constructs with mutations in the 3 CSE varied. The 3 region necessary for optimal relative survival consisted of the 3 305 nucleotides (nt), a region conserved in RUB defective-interfering RNAs, and thus this region constitutes the 3 CSE. Within the 3 CSE, deletions in the ϳ245 nt that overlap the 3 end of the E1 gene resulted in reduced relative survivals, ranging from 20 to <1% of the parental replicon survival level while most mutations within the ϳ60-nt 3 untranslated region (UTR) were lethal. None of the 3 CSE mutations affected in vitro translation of the nonstructural protein open reading frame (which is 5 proximal in the genome and encodes the enzymes involved in virus RNA replication). In cells transfected with replicons with 3 CSE mutations that survived antibiotic selection (i.e., those with mutations in the region of the 3 CSE that overlaps the E1 coding region), the amount of replicon-specific minus-strand RNA was uniform; however, the accumulation of both plus-strand RNA species, genomic and subgenomic, varied widely, indicating that this region of the RUB 3 CSE affects plus-strand RNA accumulation rather than minus-strand RNA synthesis.Rubella virus (RUB) is the sole member of the Rubivirus genus within the Togavirus family. The RUB genome consists of a single-stranded, plus-strand RNA molecule 9,762 nucleotides (nt) in length (7) which is 5Ј capped and 3Ј polyadenylated and serves as an mRNA during infection. The genome contains two open reading frames (ORFs): the 5Ј-proximal ORF encodes nonstructural proteins (NS-ORF), including the RNA-dependent RNA polymerase, that are necessary for viral RNA replication, while the 3Ј-proximal ORF encodes the viral structural proteins (SP-ORF), the capsid protein (CP), and two envelope glycoproteins, E1 and E2. The NS-ORF is translated from the genomic RNA, and the SP-ORF is translated from a subgenomic RNA. The untranslated regions (UTRs) within the RUB genome include a 40-nt sequence at the 5Ј end (5Ј UTR), a 118-nt sequence in the intragenic (junction) region between the ORFs (J-UTR), and a 59-nt sequence at the 3Ј end (3Ј UTR). Replication of the RUB genome initiates with the synthesis of a genomic-length minus-strand RNA which is then used as a template for the synthesis of both the plusstrand RNA species. The subgenomic RNA is synthesized from an internal promoter within the J-UTR.cis-Acting elements at the 3Ј end of the RUB genome (3Ј CSE) are proposed to act as promoters for minus-strand R...

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Section: Discussionmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Analysis of the 3′ cis -Acting Elements of Rubella Virus by Using Replicons Expressing a Puromycin Resistance Gene

Chen

Frolov

Icenogle

et al. 2004

J Virol

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“…Using an infectious cDNA clone containing the RUB genome, we replaced the 3Ј-proximal ORF with a number of reporter genes such as green fluorescent protein (GFP) and chloramphenicol acetyltransferase (CAT) (18). These constructs, termed replicons, replicate when introduced into cells, express the reporter gene but do not spread from cell to cell unless helper wild-type (wt) virus is present to provide the virion structural proteins.…”

Section: Diagnosis Of Rubella Virus (Rub) Infection Is Essential Aftermentioning

confidence: 99%

“…The replicon constructs RUBrep/ GFP, RUBrep/GFP-⌬NotI, RUBrep/CAT, and RUBrep/CAT-⌬NotI constructs were described previously (18,19). The plasmids were purified by CsCl isopycnic density centrifugation, followed by linearization with EcoRI (RUBrep/GFP⌬NotI) or SpeI (RUBrep/CAT and RUBrep/CAT-⌬NotI) prior to in vitro transcription, as previously described (19).…”

Section: Cells and Virusesmentioning

confidence: 99%

Novel Replicon-Based Reporter Gene Assay for Detection of Rubella Virus in Clinical Specimens

et al. 2005

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Proof of concept for a novel diagnostic assay for rubella virus (RUB) based on RUB replicons expressing reporter genes was demonstrated. RUB replicons have the structural protein coding region replaced with a reporter gene such as green fluorescent protein or chloramphenicol acetyltransferase. Previously, it was shown that a replicon construct with a specific in-frame deletion in the nonstructural protein coding region (NotI, approximately nucleotides 1500 to 2100 of the genome) failed to replicate and express the reporter gene unless rescued by a coinfecting wild-type helper RUB (W.-P. Tzeng et al., Virology 289:63-73, 2001). In the present study, it was found that rescue of reporter gene expression by NotI replicons occurred when coinfection was done with clinical specimens containing RUB, indicating that this system could be the basis for a diagnostic assay. The assay was sensitive, using laboratory RUB strains and as low a dose as one plaque-forming unit. The assay was specific in that it was positive for RUB strains of both genotypes and was negative for a panel of human viruses. It was also possible to genetically sequence the RUB present in positive clinical specimens detected in the assay for genotypic strain determination.Diagnosis of rubella virus (RUB) infection is essential after potential exposure of a woman in the first trimester of pregnancy (2), is necessary for confirmation of potential cases of congenital rubella syndrome in newborns (12), and is important in the surveillance component of rubella vaccination programs (4,11,20). Interestingly, diagnosis of RUB infection is also an important aspect of surveillance in measles vaccination programs since rubella is the most common nonmeasles rash illness screened during measles surveillance. Serodiagnostic assays for RUB infection are based on the detection of RUBspecific immunoglobulin M (IgM) in serum and saliva; IgM is also diagnostic of congenital RUB infection if present in fetal or newborn serum (1, 2). However, on the day of rash onset, only 50% of patients are IgM positive (1, 2). The percentage increases to 90% by 5 days after rash onset, and detection of RUB-specific IgG commences 3 days after rash onset; however, in light of the benign nature of the disease, it is often difficult to convince patients to return for collection of a second specimen (1, 2). In comparison, 90% of throat swab specimens taken on the day of rash onset are positive for the presence of RUB by virus isolation or reverse transcription-PCR (RT-PCR) assay (1). These techniques have also been used successfully to detect RUB in serum, saliva, nasopharyngeal washes, and urine (RT-PCR has been used to detect RUB genomes in chorionic villi or amniotic fluid in utero specimens [3,14,16,17]). Thus, direct detection of virus is more sensitive than serodiagnosis in diagnosis of RUB infection on the days immediately after presentation of symptoms.Both primary monkey kidney cell cultures or cultures of suitable continuous monkey kidney cell lines have been used to isolate RUB fro...

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Laboratory confirmation of congenital rubella syndrome in infants: An eye hospital based investigation

Rajasundari¹,

Sundaresan²,

Vijayalakshmi

et al. 2008

Journal of Medical Virology

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A total of 190 specimens from South Indian children aged 0-59 months with ocular anomalies consistent with suspected congenital rubella syndrome (CRS) were investigated. Twenty-six of the 65 infants (40%) were confirmed as CRS by detection of rubella specific IgM. Rubella RNA was detected in 41 samples from 26 infants by both real-time and block based PCR. The PCR results correlated well with the presence of anti-rubella IgM/IgG (23/27 cases with rubella IgM were PCR positive). Whereas, only 17 of 26 infants met the WHO CRS case definition. Amongst the various specimens tested from the sero-confirmed cases (n = 27), a high percentage of positives were detected in lens (92%) and oral fluid (60%) specimens, when compared to other samples. The quantification of viral load by real-time PCR demonstrated higher copy number of virus in lens samples of 0-11 months infants. The rubella viruses were characterized and revealed the circulation of genotype 2B in three South Indian states. The integrated analysis of clinical manifestations, serological and molecular data in the study has generated baseline information of rubella infection and CRS in infants with ocular anomalies.

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Rubella Virus DI RNAs and Replicons: Requirement for Nonstructural Proteins Acting in Cis for Amplification by Helper Virus

Cited by 38 publications

References 27 publications

Analysis of the 3′ cis -Acting Elements of Rubella Virus by Using Replicons Expressing a Puromycin Resistance Gene

Analysis of the 3′ cis -Acting Elements of Rubella Virus by Using Replicons Expressing a Puromycin Resistance Gene

Novel Replicon-Based Reporter Gene Assay for Detection of Rubella Virus in Clinical Specimens

Laboratory confirmation of congenital rubella syndrome in infants: An eye hospital based investigation

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