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PubMed1997

Recovery of infectious human parainfluenza virus type 3 from cDNA.

Infectious HPIV3 was produced by the intracellular coexpression of four plasmid-borne cDNAs. These separately encoded a complete HPIV3 genome (negative-sense), the HPIV3 nucleocapsid protein N, the phosphoprotein P, and the polymerase protein L. The cDNA-encoded HPIV3 genome differed from the JS wildtype (wt) strain of HPIV3 used in its construction by seven point mutations: four of these are silent mutations in the HN or L gene coding regions that serve as markers of a cDNA-derived virus, two were introduced to create an amino acid substitution that ablates an epitope recognized by the HN-specific monoclonal neutralizing antibody 423/6, and the remaining point mutation results in an incidental amino acid substitution in the HN protein at amino acid position 263. The four plasmids were transfected into HEp-2 cell monolayers and their expression was driven by T7 RNA polymerase supplied by a vaccinia virus recombinant. The titer of virus present in the harvested transfection supernatant was low (<5 PFU/ml), and the recovered recombinant virus (rJS) retained each of the seven mutations present in the cDNA from which it was derived. Despite the introduced and incidental mutations, rJS retained the wt phenotypes as regards replication at elevated temperature in vitro and efficient replication in the upper and lower respiratory tract of hamsters. rJS was also recovered from a cDNA encoding a complete antigenome (positive-sense) with slightly greater efficiency than from the negative-sense construct. The ability to generate infectious HPIV3 from cDNA should greatly enhance our ability to develop new live-attenuated parainfluenza virus vaccines, including chimeric PIV1 and PIV2 vaccines, and to understand the genetic basis of attenuation of PIV3 candidate vaccines.

Animals↗
PubMed1993

Rescue of synthetic analogs of genome RNA of human parainfluenza virus type 3.

A simple system that allows expression and packaging of a foreign gene by human parainfluenza virus type 3 (HPIV-3) has been described. First, a cDNA was constructed to encode an internally deleted version of HPIV-3 genome RNA. The viral genes were replaced with a negative sense copy of the bacterial chloramphenicol acetyl transferase (CAT) reporter gene. In vitro run-off transcription with T7 RNA polymerase synthesized an 870 nucleotide RNA that contained the antisense coding region of the CAT gene flanked by the transcription regulatory sequences and the 3' and 5' end extracistronic sequences of the HPIV-3 genome. When introduced into cells that are infected with HPIV-3, this RNA was amplified and the reporter gene was expressed, as measured by the CAT activity in the cell extract. Furthermore, the synthetic RNA was packaged into infectious virions. The addition of two extra nucleotides at the 5' end of the parental trailer region decreased the CAT activity by more than 90%, suggesting a requirement for the intact 5'-regulatory domain in the viral replicative cycle. Interestingly, the addition of one extra nucleotide to the 3' end totally abolished the CAT activity indicating that an exact 3' terminus is critical in this process.

Animals↗
PubMed1986

Dissection of RNA-primed DNA synthesis catalyzed by gene 4 protein and DNA polymerase of bacteriophage T7. Coupling of RNA primer and DNA synthesis.

Gene 4 protein and DNA polymerase of bacteriophage T7 catalyze RNA-primed DNA synthesis on single-stranded DNA templates. T7 DNA polymerase exhibits an affinity for both gene 4 protein and single-stranded DNA, and gene 4 protein binds stably to single-stranded DNA in the presence of dTTP (Nakai, H. and Richardson, C. C. (1986) J. Biol. Chem. 261, 15208-15216). Gene 4 protein-T7 DNA polymerase-template complexes may be formed in both the presence and absence of nucleoside 5'-triphosphates. The protein-template complexes may be isolated free of unbound proteins and nucleotides by gel filtration and will catalyze RNA-primed DNA synthesis in the presence of ATP, CTP, and the four deoxynucleoside 5'-triphosphates. RNA-primed DNA synthesis may be dissected into separate reactions for primer synthesis and DNA synthesis. Upon incubation of gene 4 protein with single-stranded DNA, ATP, and CTP, a primer-template complex is formed; it is likely that gene 4 protein mediates stable binding of the oligonucleotide to the template. The complex, purified free of unbound proteins and nucleotides, supports DNA synthesis upon addition of DNA polymerase and deoxynucleoside 5'-triphosphates. Association of primers with the template is increased by the presence of dTTP or DNA polymerase during primer synthesis. DNA synthesis supported by primer-template complexes initiates predominantly at gene 4 recognition sequences, indicating that primers are bound to the template at these sites.

Base Sequence↗
PubMed1991

In vivo accumulation of a turnip crinkle virus defective interfering RNA is affected by alterations in size and sequence.

Turnip crinkle virus is one of several single-stranded RNA plant viruses associated with defective interfering (DI) RNAs. A complete cDNA copy of a 344-base DI RNA (DI RNA G) was cloned downstream from a T7 RNA polymerase promoter. Transcripts synthesized in vitro were infectious when inoculated with helper virus on turnip plants. Studies of the infectivity of DI transcripts containing deletions, insertions, and single-base changes suggest that (i) in general, only the 5' two-thirds of the molecule can tolerate mutations; (ii) between 52 and 67 bases of terminal 5' sequence are required for infectivity; (iii) nucleotides in positions 68 to 138 are not specifically involved in RNA infectivity; (iv) DI RNA G molecules smaller than 327 bases are not amplified efficiently in plants.

Base Sequence↗
PubMed1991

trans rescue of a mutant poliovirus RNA polymerase function.

A series of three-nucleotide insertions was engineered into the P2 and P3 coding regions of the T7 expression plasmid pT7(tau)-PV1, which encodes a full-length copy of poliovirus type 1 (Mahoney) cDNA. When RNA derived in vitro from these mutated templates was used to transfect HeLa cells, viable virus mutants were recovered. One mutant, Sel-3D-18, which contained a single amino acid insertion in the 3Dpol coding region, was temperature sensitive for growth at 39 degrees C and showed defects in both RNA synthesis and P1 protein processing at the nonpermissive temperature. The RNA replication defect in Se1-3D-18 was identified at the level of RNA chain elongation. A highly specific and sensitive method was developed for analyzing the ability of mutant RNA templates to replicate in the presence or absence of helper functions provided in trans. This approach was used to demonstrate that RNA synthesis in Se1-3D-18 can be rescued by helper functions provided in trans.

Capsid↗
PubMed1992

Infectious Japanese encephalitis virus RNA can be synthesized from in vitro-ligated cDNA templates.

Japanese encephalitis virus (JEV) is a positive-stranded enveloped RNA virus that belongs to the family Flaviviridae. Genomic JEV RNA is approximately 11 kb long and encodes 10 proteins, 3 structural and 7 nonstructural. A full-length cDNA copy of the JEV genome was constructed by in vitro ligation of two cDNA fragments which encode the 5' (nucleotide positions 1 to 5576) and 3' (nucleotide positions 5577 to 10976) halves of the genome. T7 RNA polymerase transcripts of the ligated full-length cDNA template were infectious when transfected into BHK-21 cells. To identify the recombinant virus, a silent mutation was introduced into the clone encoding the 3' half of the genome, which abolished an XbaI site at nucleotide position 9131. Virus recovered by transfection with the transcripts contained this silent mutation, confirming its identity. Recombinant and parent viruses were identical with respect to growth and plaque production in BHK-21 cells, envelope protein expression in C6/36 cells, and neurovirulence and immunogenicity in mice. Repeated attempts to obtain infectious RNA by transcription from full-length JEV genome cDNA templates cloned into plasmid vectors were unsuccessful. Synthesis of infectious JEV RNA from in vitro-ligated JEV cDNA templates will be useful for molecular and genetic studies of flavivirus replication and virulence.

Animals↗
PubMed1970

Comparisons of F factors and R factors: existence of independent regulation groups in F factors.

The similarity of sex pili mediated by F factors and R(fi(+)) factors and the ability of R(fi(+)) factors to control by repression the functioning of pilus genes encoded by the F factor suggested that F factors and R(fi(+)) factors are closely related. Further comparisons of the episomal properties of F factors and R(fi(+)) factors, however, indicated many differences. F factors contain information for a restriction system for phages phiII and T7. Cells containing R factors are sensitive to these phages. Furthermore, R(fi(+)) factors do not repress the F factor phiII restriction system in cells containing both an R(fi(+)) factor and an F factor. R factors and F factors are heteroimmune episomes. In addition, an R(fi(+)) factor in cells containing both an R factor and an F factor does not fully repress the expression of F-factor immunity to an incoming second F factor. R-factor and F-factor replication systems are not identical. Wild-type F-factor replication genes will complement the mutant F(ts114)lac(+) replication genes in cells containing two F factors. The F(ts114)lac(+) episome is retained when these cells are grown at 42 C; however, cells containing an R(fi(+)) factor and F(ts114)lac(+) lose the F(ts114)lac(+) when grown at 42 C, at the same rate as cells containing only the F(ts114)lac(+). The replication system of the R(fi(+)) factor will not complement the mutant F(ts114)lac(+) replication system.

Bacteriophages↗
PubMed1993

Deletion mapping of a mouse hepatitis virus defective interfering RNA reveals the requirement of an internal and discontiguous sequence for replication.

All of the defective interfering (DI) RNAs of mouse hepatitis virus (MHV) contain both the 5' and 3' ends of the viral genomic RNA, which presumably include the cis sequences required for RNA replication. To define the replication signal of MHV RNA, we have used a vaccinia virus-T7 polymerase-transcribed MHV DI RNA to study the effects of sequence deletion on DI RNA replication. Following infection of susceptible cells with a recombinant vaccinia virus expressing T7 RNA polymerase, various cDNA clones derived from a DI RNA (DIssF) of the JHM strain of MHV, which is a 3.5-kb naturally occurring DI RNA, behind a T7 promoter were transfected. On superinfection with a helper MHV, the ability of various DI RNAs to replicate was determined. Serial deletions from the middle of the RNA toward both the 5' and 3' ends demonstrated that 859 nucleotides from the 5' end and 436 nucleotides from the 3' end of the MHV RNA genome were necessary for RNA replication. Surprisingly, an additional stretch of 135 nucleotides located at 3.1 to 3.3 kb from the 5' end of the genome was also required. This stretch is discontiguous from the 5'-end cis replication signal and is present in all of the naturally occurring DI RNAs studied so far. The requirement for a long stretch of 5'- and 3'-end sequences predicts that the subgenomic MHV mRNAs cannot replicate. The efficiency of RNA replication varied with different cDNA constructs, suggesting possible interaction between different regions of DI RNA. The identification of MHV RNA replication signals allowed the construction of an MHV DI-based expression vector, which can express foreign genes, such as the chloramphenicol acetyltransferase gene.

Animals↗
PubMed1997

Translation and replication properties of the human rhinovirus genome in vivo and in vitro.

The poor translation efficiency of genome-length human rhinovirus RNA in vitro using HeLa cell extract-supplemented rabbit reticulocyte lysate has hampered the study of rhinovirus IRES-mediated translation and polyprotein synthesis in a cell-free system. In contrast, the efficient in vitro translation characteristics of poliovirus RNAs have ultimately allowed the programming of cell-free coupled translation/replication extracts which are able to produce infectious poliovirus particles in vitro. A possible explanation for the decreased burst size observed during the course of a rhinovirus infection, compared to poliovirus infection, is reduced levels of polyprotein synthesis in vivo. In order to test this hypothesis and extend in vitro translation/replication technology to the study of human rhinoviruses, a chimeric cDNA construct was engineered which allowed the in vitro synthesis of T7 transcripts containing the intact poliovirus type 1 (PV1) 5' noncoding region (5' NCR) and initiation codon upstream of the human rhinovirus 14 (HRV14) polyprotein-coding region and 3'-terminal sequences. These chimeric RNAs translated efficiently in vitro and were used successfully to program a cell-free replication extract. Unexpectedly, parental HRV14 RNAs also translated efficiently in the HeLa cell-free translation/replication extract but replicated less efficiently than the chimera in vitro. The chimeric HRV14/PV1 RNAs were infectious and gave rise to a virus with a growth phenotype similar to that of parental HRV14. Preliminary characterization of this chimeric virus suggests that the biological properties characteristic of rhinovirus in vivo are determined primarily by the rhinovirus gene products. Although the translation efficiency of the HRV14 5' NCR may be a limitation in rabbit reticulocyte lysate-based in vitro translation extracts, it does not appear to be a major limiting determinant for growth of rhinovirus in vivo or replication in the HeLa cell-free extract.

Animals↗
PubMed1991

Strand-specific detection of enteroviral RNA in myocardial tissue by in situ hybridization.

In this report we describe the development and application of single-stranded RNA probes for strand-specific detection of enterovirus RNA in infected heart tissue by in situ hybridization. For synthesis of RNA probes a full-length reverse-transcribed, recombinant CVB3 cDNA was inserted into the transcription vector pSPT18. Run-off transcripts of plus-strand and minus-strand orientation were produced using either T7 or SP6 RNA polymerase. Binding specificity and sensitivity of the radioactively labelled RNA probes were determined by slot-blot hybridization. Due to the high degree of genetic identity among enteroviruses, the in vitro transcribed CVB3 RNA probes hybridized with various enterovirus serotypes, including group A and B coxsackieviruses and echoviruses, which are commonly implicated in human viral heart disease. Strand-specific in situ hybridization led to detection of viral plus-strand or minus-strand RNA in infected cell cultures and in myocardial tissue sections of infected mice. In consecutive sections either viral genomic plus-strand RNA or complementary minus-strand RNA were localized in the same infected myocardial cells. In situ hybridization with enterovirus-specific and highly sensitive single-stranded RNA probes is of particular interest for the diagnosis of myocardial infections and for studies concerning viral RNA replication.

Animals↗
PubMed2004

Recovery of human metapneumovirus genetic lineages a and B from cloned cDNA.

Human metapneumovirus (hMPV) is a newly discovered pathogen associated with respiratory tract illness, primarily in young children, immunocompromised individuals, and the elderly. The genomic sequence of the prototype hMPV isolate NL/1/00 without the terminal leader and trailer sequences has been reported previously. Here we describe the leader and trailer sequences of two hMPV isolates, NL/1/00 and NL/1/99, representing the two main genetic lineages of hMPV. Minigenome constructs in which the green fluorescent protein or chloramphenicol acetyltransferase genes are flanked by the viral genomic ends derived from both hMPV lineages and transcribed using a T7 RNA polymerase promoter-terminator cassette were generated. Cotransfection of minigenome constructs with plasmids expressing the polymerase complex components L, P, N, and M2.1 in 293T or baby hamster kidney cells resulted in expression of the reporter genes. When the minigenome was replaced by a sense or antisense full-length cDNA copy of the NL/1/00 or NL/1/99 viral genomes, recombinant virus was recovered from transfected cells. Viral titers up to 10(7.2) and 10(5.7) 50% tissue culture infective dose/ml were achieved with the sense and antisense plasmids, respectively. The recombinant viruses replicated with kinetics similar to those of the parental viruses in Vero cells. This reverse genetics system provides an important new tool for applied and fundamental research.

5' Untranslated Regions↗
PubMed1998

The ability of a variety of polymerases to synthesize past site-specific cis-syn, trans-syn-II, (6-4), and Dewar photoproducts of thymidylyl-(3'-->5')-thymidine.

The role of photoproduct structure, 3' --> 5' exonuclease activity, and processivity on polynucleotide synthesis past photoproducts of thymidylyl-(3' --> 5')-thymidine was investigated. Both Moloney murine leukemia virus reverse transcriptase and 3' --> 5' exonuclease-deficient (exo-) Vent polymerase were blocked by all photoproducts, whereas Taq polymerase could slowly bypass the cis-syn dimer. T7 RNA polymerase was able to bypass all the photoproducts in the order cis-syn > Dewar > (6-4) > trans-syn-II. Klenow fragment could not bypass any of the photoproducts, but an exo- mutant could bypass the cis-syn dimer to a greater extent than the others. Likewise T7 DNA polymerase, composed of the T7 gene 5 protein and Escherichia coli thioredoxin, was blocked by all the photoproducts, but the exo- mutant Sequenase 2.0 was able to bypass them all in the order cis-syn > Dewar > trans-syn-II > (6-4). No bypass occurred with an exo- gene 5 protein in the absence of the thioredoxin processivity factor. Bypass of the cis-syn and trans-syn-II products by Sequenase 2.0 was essentially non-mutagenic, whereas about 20% dTMP was inserted opposite the 5'-T of the Dewar photoproduct. A mechanism involving a transient abasic site is proposed to account for the preferential incorporation of dAMP opposite the 3'-T of the photoproducts.

Base Sequence↗
PubMed1995

RNA transcripts derived from a cloned full-length copy of the feline calicivirus genome do not require VpG for infectivity.

Feline calicivirus (FCV) is a positive-strand, nonenveloped RNA virus in the family Caliciviridae. A cDNA library of the Urbana (URB) strain of FCV was generated and the sequence of the genome was determined from overlapping clones except for 13 bases from the 5'-end. The 5'-end sequence was identified by analysis of clones derived by RT-PCR across the ligated 5'- and 3'-ends of the RNA genome. A full-length cDNA clone of the RNA genome of the URB strain was constructed and placed downstream of the T7 RNA polymerase promoter and RNA transcripts generated in vitro from this clone were infectious when introduced into feline kidney cells. A virus-encoded genome-linked protein, VpG, which is considered to be essential for infectivity of wild-type genomic FCV RNA, was not required for the initiation of FCV infection by the synthetic transcripts. However, the addition of a cap structure analog (m7G(5')ppp(5')G) during in vitro transcription of the synthetic RNA was necessary for successful virus recovery. Two silent mutations engineered into the full-length clone were identified in the genomic RNA from recovered progeny virus. This system of introducing site-specific genetic changes into the genome of feline calicivirus and the recovery of infectious mutant viruses will enable studies related to the molecular basis for replication, growth restriction, and pathogenicity of this and other members of the Caliciviridae.

Animals↗
PubMed1990

Infectious in vitro transcripts from a plum pox potyvirus cDNA clone.

A full-length cDNA clone of the 9786 nt plum pox virus (PPV) RNA genome has been cloned downstream from a phage T7 RNA polymerase promoter. The RNAs synthesized by in vitro run-off transcription in the presence of the 5' cap analog m7GpppG were infectious in Nicotiana clevelandii plants. No infectivity was detected when the transcriptions were carried out in the absence of the cap analog. Inoculations of the local lesion host Chenopodium foetidum indicated that the infectivity of the synthetic transcripts was about 1% of that of the native viral RNA. An extra G present at the 5' terminus of the transcripts was lost during their replication in plants, and the typical length distribution of the poly(A) tails was recovered. The viral RNA recovered from transcript-infected plants had approximately the same specific infectivity as native viral RNA. A G/A sequence heterogeneity found between different cDNA subgenomic clones was used to demonstrate that the infections were caused by the in vitro transcripts and were not the result of contamination.

Base Sequence↗
PubMed1995

Induction of membrane proliferation by poliovirus proteins 2C and 2BC.

Poliovirus infection leads to the appearance of a number of cytoplasmic vacuoles involved in the replication of virus genomes. To characterize the viral proteins involved in membrane proliferation different poliovirus proteins have been expressed in HeLa cells. Two recombinant vaccinia viruses have been obtained that express poliovirus protein 2C, one under the 5' untranslated (UTR) sequence of poliovirus and another under the leader region of EMC virus. Expression of 2C was very efficient in both cases, although better results were obtained when poliovirus 2C was expressed under the 5'UTR sequence of EMC virus. Transient expression of poliovirus proteins 2B, 2C or 2BC placed under a T7 promoter was analyzed using a recombinant vaccinia virus that contains the bacteriophage T7 RNA polymerase. The expression of 2C, or 2BC, contrary to 2B, was able to induce the proliferation of vacuoles morphologically similar to those found during poliovirus infection. These findings indicate that poliovirus protein 2C, in addition to its NTPase and RNA binding activities, is also endowed with the capacity to induce the formation of cytoplasmic vacuoles.

Animals↗
PubMed1979

Characterization of the ribonucleic acid primers and the deoxyribonucleic acid product synthesized by the DNA polymerase and gene 4 protein of bacteriophage T7.

The DNA polymerase and gene 4 protein of phage T7, in the presence of helix-destabilizing protein (DNA binding protein), catalyze DNA synthesis on duplex templates. As has been previously shown (Kolodner, R. D., and Richardson, C. C. (1978) 4. Biol. Chem. 253, 574-584), in the absence of ribonucleoside 5'-triphosphates DNA synthesis is initiated at nicks, and all of the newly synthesized DNA is covalently attached to the template. In this paper we characterize the DNA synthesized in the presence of ribonucleoside 5'-triphophates and show that, in contrast, the major portion of the newly synthesized DNA is not attached to the template, having an average chain length of 5000 to 6000 nucleotides. In addition, each chain of newly synthesized DNA is terminated at its 5'-end by a covalently attached tetranucleotide RNA primer whose sequence is predominantly pppApCpCpC and pppApCpCpA. The results of isotope transfer experiments are in agreement with the number of initiation events determined by the incorporation of [gamma-32P]ATP and indicate that each of the four deoxyribonucleotides is present at the RNA-DNA junction.

DNA Helicases↗
PubMed1986

Synthesis of infectious poliovirus RNA by purified T7 RNA polymerase.

Plasmids containing the entire cDNA sequence of poliovirus type 1 (Mahoney strain) under control of a promoter for T7 RNA polymerase have been constructed. Purified T7 RNA polymerase efficiently transcribes the entire poliovirus cDNA in either direction to produce full-length poliovirus RNA [(+)RNA] or its complement [(-)RNA]. The (+)RNA produced initially had 60 nucleotides on the 5' side of the poliovirus RNA sequence, including a string of 18 consecutive guanine residues generated in the original cloning and an additional 626 nucleotides of pBR322 sequence beyond the poly(A) tract at the 3' end. Such RNA, while much more infectious than the plasmid DNA, is only about 0.1% as infectious as RNA isolated from the virus. Subsequently, a T7 promoter was placed only 2 base pairs ahead of the poliovirus sequence, so that T7 RNA polymerase synthesizes poliovirus RNA with only 2 additional guanine residues at the 5' end and no more than seven nucleotides past the poly(A) tract at the 3' end. Such RNA has much higher specific infectivity, about 5% that of RNA isolated from the virus. The ability to make infectious poliovirus RNA efficiently from cloned DNA makes it possible to apply techniques of in vitro mutagenesis to the analysis of poliovirus functions and the construction of novel and perhaps useful derivatives of poliovirus. A source of variant RNAs should also allow detailed study of the synthesis and processing of poliovirus proteins in vitro.

Cloning, Molecular↗
PubMed1996

Initiation of Sendai virus multiplication from transfected cDNA or RNA with negative or positive sense.

BACKGROUND: The mononegavirus superfamily (Mononegavirales) comprises three families, Rhabdoviridae, Paramyxoviridae and Filoviridae. These viruses possess a single stranded negative sense RNA as the genome. Recent success in the recovery of infectious virus from a transfected cDNA of mononegaviruses including Sendai virus, a prototypic paramyxovirus, is opening the possibility of their genetic engineering. However, infectious viruses have been recovered only by initiating the infectious cycle with cDNA directing the synthesis of antigenomic positive sense (+)RNA. Starting with genomic negative sense (-)RNA has been unsuccessful. Furthermore, the recovery efficiency has often been extremely low. RESULTS: We describe here an analogous system that allows recovery of Sendai virus at a high rate, from cells in which the transfected cDNA and plasmids to support the synthesis of viral nucleocapsid protein and RNA polymerases are coexpressed by vaccinia virus-driven bacteriophage T7 polymerase. Our system was able to recover the virus from cDNA directing not only (+)RNA but also (-)RNA. Moreover, using this system, we succeeded in recovery of the virus by transfection of in vitro synthesized (+)RNA or (-)RNA. This improved virus recovery appeared to be accomplished by supplying the supporting plasmids at an optimal ratio and by minimizing the cytopathic effect of the vaccinia virus by specific inhibitors. In addition, it was probably critical that our cDNAs were constructed to generate viral authentic RNAs without adding T7 promoter-specific nucleotides to the 5' ends. An immediate application of the system was demonstrated by the creation of a candidate vaccine strain with a predetermined attenuating mutation in the cleavage-activation site of the viral fusion glycoprotein. CONCLUSION: We have established methods which greatly improve the recovery of Sendai virus from cDNA. There is essentially no absolute obstacle to recovery of the virus from the (-)RNA template. Even the complete full length RNA chain in the naked form appears to be properly encapsidated to become a functional template.

Amino Acid Sequence↗