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At least 37 records · Page 2Linked to original sources
PubMed1989

Augmentation of helper virus replication in the presence of defective retrovirus.

The interaction between defective spleen focus-forming virus (SFFV) and helper virus(es) in Friend virus (FV) complex has been assumed to be one-way, with the helper virus complementing SFFV by supplying necessary virion components. To test this assumption the expression of both SFFV and helper virus in partially congenic mice which differ at the Fv-2 locus, a gene that specifically controls susceptibility to SFFV, was analysed. When the mice were infected with LLV (a strain of Friend SFFV-free helper virus), there was no detectable effect of Fv-2 genotype on LLV expression as tested by virus infectivity in the XC plaque assay or by quantitative viral antigen analysis in an immunoprecipitation assay. However, after infection with FV complex there was an amplification of LLV (as well as SFFV) synthesis in Fv-2s as compared with Fv-2r hosts. To determine whether the increased LLV synthesis in Fv-2s mice was due to an increased population of susceptible target cells as a result of SFFV infection and/or transformation, the ratios of LLV-infected cells in the spleens of LLV- and FV-infected Fv-2s hosts in an infectious centre assay, were compared. Since the percentage of LLV-infected cells was equivalent in both instances, the higher rate of LLV synthesis after infection with FV complex was presumably due to intrinsic properties of SFFV-infected erythroid cells.

Animals↗
PubMed1983

Structure of the FBJ murine osteosarcoma virus genome: molecular cloning of its associated helper virus and the cellular homolog of the v-fos gene from mouse and human cells.

The 8.2-kilobase (kb) unintegrated circular DNA form of the FBJ murine leukemia virus (FBJ-MLV) was linearized by cleavage at the single HindIII site, molecularly cloned into bacteriophage Charon 30, and subsequently subcloned into pBR322 (pFBJ-MLV-1). Both FBJ-MLV virion RNA and pFBJ-MLV-1 DNA were used to investigate the arrangement of helper virus sequences in the FBJ murine osteosarcoma virus genome (FBJ-MSV) by heteroduplex formation with cloned FBJ-MSV proviral DNA. The results showed that the FBJ-MSV genome contained 0.8 kb of helper virus sequence at its 5' terminus and 0.98 kb at its 3' terminus. Approximately 6.8 kb of helper virus sequence had been deleted, and 1.7 kb of unrelated sequence was inserted into the FBJ-MSV genome. This substituted region contains v-fos, the transforming gene of FBJ-MSV. Using a probe specific for v-fos, we have cloned homologous sequences (c-fos) from mouse and human chromosomal DNA. Heteroduplex analysis of FBJ-MSV DNA with these recombinant clones showed that both the c-fos(mouse) and the c-fos(human) sequences hybridized to the entire 1.7-kb v-fos region. However, five regions of homology of 0.27, 0.26, 0.14, 0.5, and 0.5 kb were separated by four regions of nonhomology of 0.76, 0.55, 0.1, and 0.1 kb from 5' to 3' with respect to the FBJ-MSV genome. The size of these sequences showed striking similarity in both c-fos(mouse) and c-fos(human).

Animals↗
PubMed1982

Rauscher spleen focus-forming virus: biological properties and relationship to helper viruses.

A Rauscher virus (RV)-transformed erythroid cell line, RA-1, was shown to be a non-producer cell line. RA-1 cells express not only gp51-54 env-related glycoprotein, but also gp70, which is more closely related to gp51-54 coded by a recombinant env gene than to the MuLV-R gp70. RA-1 cells could be infected by Friend, Moloney and Gross viruses, but not by the homologous Rauscher murine leukaemia virus. Rescue of spleen focus-forming activity was obtained on infection of these cells with MuLV-F or MuLV-Mol, but not with MuLV-Gross. The RNA of the RV complex resembles closely that of Friend virus (FV). It contains a 32S, presumably defective, genome, which most likely is responsible for spleen focus formation, and a 35S helper virus genome. Oligonucleotide fingerprint data suggest that RV has evolved independently of FV. Erythroid early BFU-E cells of mice infected with RV of Friend helper virus-infected RA-1 cells were shown to require no addition of conditioned medium to form large erythroid colonies (BFU-E) in the presence of only small amounts of erythropoietin.

Animals↗
PubMed2001

Transient expression of a GUS reporter gene from cauliflower mosaic virus replacement vectors in the presence and absence of helper virus.

Vectors based upon the genome of cauliflower mosaic virus (CaMV) have only a limited capacity for replicating foreign DNA in plants. A helper virus system has been developed to complement CaMV constructs capable of carrying a large foreign gene (glucuronidase; GUS). GUS replaced part or all of the non-essential CaMV gene II and the essential genes III, IV and V. This construct was co-inoculated mechanically with wild-type CaMV helper virus onto Brassica rapa leaves to promote GUS vector complementation. After 1 week, blue foci of GUS activity were observed in the centres of the local lesions. Leaves inoculated with the GUS construct in the absence of helper virus showed randomly distributed foci of GUS activity that were generally smaller than the lesion-associated GUS foci. Inoculation with a simple non-replicating CaMV 35S promoter-GUS construct also produced small GUS foci. Co-inoculation of helper virus with CaMV gene replacement vectors in which replication was prevented by moving the primer-binding site or by deletion of an essential splice acceptor produced only small, randomly distributed GUS activity foci, demonstrating that the lesion-associated foci were produced by gene expression from replicating constructs. These experiments show that CaMV genes III-V can be complemented by wild-type virus and replacement gene vectors can be used for transient gene expression studies with CaMV constructs that distinguish gene expression associated with a replicating vector from that associated with a non-replicating vector.

Amino Acid Sequence↗
PubMed1997

Human genital tissues containing DNA of adeno-associated virus lack DNA sequences of the helper viruses adenovirus, herpes simplex virus or cytomegalovirus but frequently contain human papillomavirus DNA.

The detection of DNA of the helper virus-dependent adeno-associated virus type 2 (AAV-2) in biopsies of material from spontaneous abortion and in tissue samples from the uterus raises the question of whether sequences of known helper viruses can be detected simultaneously within the same specimen despite the lack of histological evidence for the presence of lytic viruses. Therefore, we performed PCR analyses with primers detecting DNA sequences of viruses (adenovirus, herpes simplex virus and human cytomegalovirus) known for their helper activity in the replication of adeno-associated viruses. In addition, PCR was performed to detect DNA of human papillomaviruses (HPV), which were recently shown to be able to help AAV replication in vitro. In no cases were sequences of the known helper viruses found. However, HPV DNA was detected in approximately 60% of paraffin sections from uterus biopsies and cervical lesions containing AAV DNA and in approximately 70% of material from early miscarriage. This finding suggests that HPV may be a helper virus for AAV.

Abortion, Spontaneous↗
PubMed1998

Adeno-associated virus vectors can be efficiently produced without helper virus.

The purpose of this work was to develop an efficient method for the production of adeno-associated virus (AAV) vectors in the absence of helper virus. The adenovirus regions that mediate AAV vector replication were identified and assembled into a helper plasmid. These included the VA, E2A and E4 regions. When this helper plasmid was cotransfected into 293 cells, along with plasmids encoding the AAV vector, and rep and cap genes, AAV vector was produced as efficiently as when using adenovirus infection as a source of help. CMV-driven constructs expressing the E4orf6 and the 72-M(r), E2A proteins were able to functionally replace the E4 and E2A regions, respectively. Therefore the minimum set of genes required to produce AAV helper activity equivalent to that provided by adenovirus infection consists of, or is a subset of, the following genes: the E4orf6 gene, the 72-M(r), E2A protein gene, the VA RNA genes and the E1 region. AAV vector preparations made with adenovirus and by the helper virus-free method were essentially indistinguishable with respect to particle density, particle to infectivity ratio, capsimer ratio and efficiency of muscle transduction in vivo. Only AAV vector preparations made by the helper virus-free method were not reactive with anti-adenovirus sera.

Adenoviridae↗
PubMed1981

Molecular cloning and comparative analyses of the genomes of simian sarcoma virus and its associated helper virus.

Closed circular viral DNA of simian sarcoma virus (SSV) and simian sarcoma-associated virus (SSAV) obtained from acutely infected dog cells was purified on preparative agarose gels, cleaved with EcoRI, and cloned in the phage lambda vector Charon 21A. The cloned 9-kilobase SSAV genome (B11) has the same restriction map as the bulk of the unintegrated linear SSAV DNA intermediate. Heteroduplex analysis between an SSV clone (lambda-C60) and an SSAV clone (lambda-B11) showed two substitution loops and one deletion loop. By using detailed restriction enzyme mapping and electron microscopic analysis, we showed that one of the substitution loops corresponds to an inversion of one of the two long terminal repeat units and adjacent cellular sequences in C60. The other substitution loop mapped close to the 3' long terminal repeat. At least part of this region was shown to contain SSV-specific sequences not shared by SSAV. The 1.9-kilobase deletion mapped at 3.5-5.5 kilobases of the linear SSAV genome, corresponding to most, if not all, of the pol gene.

Chromosome Inversion↗
PubMed1977

Replication of viral RNA by a defective interfering vesicular stomatitis virus particle in the absence of helper virus.

The genome of a defective interfering particle (DILT) derived from the heat-resistant strain of vesicular stomatitis virus is expressed in vivo without the assistance of infectious helper virus. The rates of RNA synthesis in the presence of cycloheximide (primary transcription) are the same when infections are with equal numbers of physical particles of DILT or virus. With this treatment, DILT synthesizes only 12-17S mRNAs as characterized by size, polarity, and polyadenylylation. In the absence of cycloheximide, DILT-infected cells produce not only these mRNAs but also a 28S RNA species. This RNA, which represents one half of the viral specific RNA, contains newly synthesized full-length (+) and (-) strand DILT RNA. Both strands are found intracellularly as ribonucleoprotein complexes. Without cycloheximide present, the rate of RNA synthesis by DILT was less than that by virus. This curtailment is most likely due to the inability of DILT to synthesize L protein mRNA. An expanded role for defective interfering particles in infection is discussed.

Cells, Cultured↗
PubMedDate not supplied

Production of helper-dependent adenovirus vector relies on helper virus structure and complementing.

BACKGROUND: The helper-dependent (HD) adenoviral (Ad) vector relies on a helper virus to provide viral proteins for vector amplification. HD-Ad vectors can significantly increase therapeutic gene expression and improve safety. However, the yield of an HD-Ad vector is generally lower than that of an E1-deleted first-generation vector, likely due to the alterations in viral E3 or packaging regions of a helper virus that attenuate its replication and complementing for an HD-Ad vector. METHODS: To study this question and improve HD-Ad vector production, we have generated four different helper viruses with a wild-type or deleted E3 region, and with a relocated loxP. We have also constructed a first-generation vector with a wild-type E3 region and without the loxP site. We compared the replication of these viruses in Cre-positive and -negative cells and studied their complementing for HD-Ad vector production. RESULTS: Viruses with deleted E3 formed smaller plaques and produced lower titer compared with viruses containing the E3 region. The site where a loxP is inserted can also affect virus replication. Higher yield of HD-Ad vector was obtained when a helper virus with wild-type E3 was used. We also showed that deletion of the packaging signal in a helper virus through loxP/Cre interaction decreased the viral DNA complementing ability. CONCLUSIONS: Although the E3 region is not essential for adenovirus replication in vivo, deletion of this region attenuates virus replication. Production of HD-Ad vector can be further improved by modifications in helper virus structure.

Adenoviridae↗
PubMed1986

The virulent satellite RNA of turnip crinkle virus has a major domain homologous to the 3' end of the helper virus genome.

RNA C (355 bases), RNA D (194 bases) and RNA F (230 bases) are small, linear satellite RNAs of turnip crinkle virus (TCV) which have been cloned as cDNAs and sequenced in this study. These RNAs produce dramatically different disease symptoms in infected plants. RNA C is a virulent satellite that intensifies virus symptoms when co-inoculated with its helper virus in turnip plants, while RNA D and RNA F are avirulent. RNA D and RNA F, the avirulent satellites, are closely related to each other except that RNA F has a 36-base insert near its 3' end, not found in RNA D. The 189 bases at the 5' end of RNA C, the virulent satellite, are homologous to the entire sequence of RNA D. However, the 3' half of RNA C, is composed of 166 bases which are nearly identical to two regions at the 3' end of the TCV helper virus genome. Hence, the virulent satellite is a composite molecule with one domain at its 5' end homologous to the other avirulent satellites and another domain at its 3' end homologous to the helper virus genome. All four TCV RNAs, RNAs C, D and F and the helper virus genome have identical 7 bases at their 3' ends. The secondary structure of RNA C deduced from the sequence can be folded into two separate domains - the domain of helper virus genome homology and the domain homologous to other TCV satellite RNAs. Comparative sequences of several different RNA C clones reveal that this satellite is a population of molecules with sequence and length heterogeneity.

Journal Article↗
PubMedDate not supplied

Influence of the helper virus on expression of beta-galactosidase from a defective HSV-1 vector, pHSVlac.

Defective herpes simplex virus type 1 (HSV-1) vectors can deliver genes into both mitotic and postmitotic cells, including neurons and these vectors, therefore, have great potential use. pHSVlac, the prototype HSV-1 vector, expresses the E. coli Lac Z gene from the HSV-1 immediate early 4/5 promoter. pHSVlac can stably express beta-galactosidase in a range of mammalian cell lines and in neurons from throughout the nervous system, both in culture and in the adult rat brain. Thus, HSV-1 vectors may be useful for studying HSV-1 latency, neuronal physiology, and performing gene therapy for neurological conditions. A virus stock of pHSVlac consists of identical HSV-1 particles containing either pHSVlac DNA or the HSV-1 helper virus DNA. Thus, a cell can be infected with the pHSVlac virus, the helper virus, or both; consequently, it is important to determine if the helper virus influences the behavior of pHSVlac. The effect of the helper virus on expression of beta-galactosidase from pHSVlac was investigated: it was demonstrated first, that pHSVlac can be efficiently packaged into HSV-1 virus particles using each of five HSV-1 temperature sensitive (ts) mutants as helper virus. Second, pHSVlac grown with each of the five HSV-1 ts mutants expressed high levels of beta-galactosidase. Third, pHSVlac grown with HSV-1 strain 17 ts K as helper virus expresses the same amount of beta-galactosidase in the absence or presence of ts K. Thus, pHSVlac can efficiently express a gene independent of the HSV-1 helper virus.

Adrenal Gland Neoplasms↗
PubMed1998

Deletion of internal sequences results in tobacco mosaic virus defective RNAs that accumulate to high levels without interfering with replication of the helper virus.

Deletion of certain internal sequences of the tobacco mosaic tobamovirus (TMV) genome was required to create replication-defective RNAs (dRNA) that were replicated in trans by TMV. All dRNAs that accumulated to detectable levels were missing nucleotides 3420-4902, which appeared to constitute a core region that inhibited replication in trans. Deletion of additional sequences resulted in dRNAs that varied tremendously in ability to be replicated from none to levels exceeding that of the helper viral RNA. Accumulation of dRNA negative- and positive-stranded RNAs of each dRNA paralleled those of the helper virus. Negative-stranded RNA accumulation of both helper and dRNA ceased at the same early time in the infection while synthesis of both positive-stranded RNAs continued, suggesting that both dRNAs and helper virus RNAs were synthesized from the same pool of replicase complexes. Positive- to negative-stranded RNA ratios for the dRNAs were similar to, or slightly greater than the wild-type helper virus. Full-length dRNAs were not supported in trans by a replication-competent helper virus. Even though some of the artificially constructed dRNAs accumulated to levels exceeding the level of the helper virus, none appreciably affected the replication of the helper virus, suggesting that the dRNAs are produced from "excess" replicase capacity.

DNA-Directed DNA Polymerase↗
PubMed1995

Effect of temperature on cucumber mosaic virus satellite-induced lethal tomato necrosis is helper virus strain dependent.

The effect of temperature on the response of tomato (Lycopersicon esculentum Mill. cv. Rutgers) to infections with the necrogenic cucumber mosaic virus (CMV) satellite D-CARNA 5 was investigated with each of four CMV strains D, 1, Y and S functioning as helper virus. At 24 degrees C lethal necrosis was observed in all infections. However, at 32 degrees C the response varied from total absence or reduction of necrosis with some strains to accelerated lethal necrosis with others. The total lack of necrotic response with CMV-S and the aggravated necrosis with CMV-Y at the higher temperature both turned out to be independent of the coinfecting satellite, and rather to correlate with the changing rate of viral RNA accumulation in tomato, which probably was responsible for the changes in pathogenic response. However, when CMV-D was helper virus, satellite accumulation decreased, while with CMV-1 it increased, respectively, while viral RNA accumulations were not seriously affected. Although these profound effects of temperature seem to link the necrotic response of tomato to the competitive replication dynamics of the infecting virus/satellite combination in the case of CMV-D/D-CARNA 5, temperature effects at other levels of disease induction probably play an important role as well.

Cucumovirus↗
PubMed2000

Chimeric retroviral helper virus and picornavirus IRES sequence to eliminate DNA methylation for improved retroviral packaging cells.

Most retroviral packaging cell lines were established by a helper virus plasmid cotransfected with a separate plasmid encoding a selection marker. Since this selection marker coexisted in trans with the helper virus sequence, helper virus gene expression could be inactivated by host DNA methylation despite selection for the cotransfected selection marker. We have reported that DNA methylation could occur in the long terminal repeat (LTR) region of helper virus in vector producer cells (VPC) in up to 2% of the population per day (W. B. Young, G. L. Lindberg, and C. J. Link, Jr., J. Virol. 74:3177-3187, 2000). To overcome host cell DNA methylation that suppresses viral gene expression, we constructed a chimeric retroviral helper virus, pAM3-IRES-Zeo, that contains Moloney murine leukemia virus as a helper virus and a picornavirus internal ribosome entry site (IRES) sequence followed by a Zeocin selection marker at the 3' end of the env sequence. This pAM3-IRES-Zeo permitted selection for intact and functional helper virus in transfected cells without subcloning. By selection with Zeocin, a mixed population of pAM3-IRES-Zeo-transfected NIH3T3 cells (AMIZ cells) was maintained with little or no DNA methylation of the helper virus 5' LTR. The high level of pAM3-IRES-Zeo gene expression resulted in no detectable vector superinfection and in high vector titers (2 x 10(6) to 1.5 x 10(7) CFU/ml) after introduction of a retroviral vector. When Zeocin selection was withdrawn from AMIZ cells, methylation of the 5' LTR increased from 17 to 36% of the population during 67 days of continuous culture and the cells became susceptible to superinfection. During this period, gene expression of pAM3-IRES-Zeo decreased and vector titer production was reduced to 2 x 10(4) CFU/ml. These data demonstrate an important role of DNA methylation in the genetic instability of VPC. The chimeric helper virus allows the establishment of a mixed population of packaging cells capable of high-level and sustained vector production without cloning procedures.

3T3 Cells↗
PubMed2000

Characterization of influenza virus NS1 protein by using a novel helper-virus-free reverse genetic system.

We have developed a novel helper-virus-free reverse genetic system to genetically manipulate influenza A viruses. The RNPs, which were purified from the influenza A/WSN/33 (WSN) virus, were treated with RNase H in the presence of NS (nonstructural) cDNA fragments. This specifically digested the NS RNP. The NS-digested RNPs thus obtained were transfected into cells together with the in vitro-reconstituted NS RNP. The NS-digested RNPs alone did not rescue viruses; however, cotransfection with the NS RNP did. This protocol was also used to rescue the NP transfectant. We obtained two NS1 mutants, dl12 and N110, using this protocol. The dl12 NS gene contains a deletion of 12 amino acids at positions 66 to 77 near the N terminus. This virus was temperature sensitive in Madin-Darby bovine kidney (MDBK) cells as well as in Vero cells. The translation of all viral proteins as well as cellular proteins was significantly disrupted during a later time of infection at the nonpermissive temperature of 39 degrees C. The N110 mutant consists of 110 amino acids which are the N-terminal 48% of the WSN virus NS1 protein. Growth of this virus was significantly reduced at any temperature. In the virus-infected cells, translation of the M1 protein was reduced to 10 to 20% of that of the wild-type virus; however, the translation of neither the nucleoprotein nor NS1 was significantly interfered with, indicating the important role of NS1 in translational stimulation of the M1 protein.

Animals↗
PubMedDate not supplied

Cucumber-mosaic-virus-associated RNA-5. XII. Symptom-modulating effect is codetermined by the helper virus satellite replication support function.

In tomato, the disease-modulating effects of a cucumber mosaic virus (CMV) satellite isolate from Belgium, here designated T-CARNA-5 (CARNA-5 = CMV-associated RNA-5), were found to be different depending on the supporting helper virus strain. With two CMV strains, T-CARNA-5 induced lethal necrosis, but with a third strain from Ixora spp. (CMV-Ix), aggravated stunting was observed. However, the primary structure of the T-CARNA-5 contained within virus isolated from tobacco or tomato infected with each of these three CMV strains, conformed to the conserved sequence profile of CARNA-5 isolates which are necrogenic in tomato. Dilution endpoint bioassay of T-CARNA-5 established a direct cause-effect relationship between it and tomato necrosis or stunting, depending on the helper virus. Total nucleic acid extracts taken at different times from tomato plants infected with the above CMV strains and T- or S-CARNA-5 (used as non-necrogenic control) showed viral RNA, ssCARNA-5 and dsCARNA-5 to be present in significant amounts, but in sometimes dissimilar proportions depending on the combination; except in CMV-Ix/S-CARNA-5 infection where neither ss-nor dsCARNA-5 was found. The experiments established that CARNA-5 biological expression studies in CMV-infected tomato have to take into account the helper virus satellite replication support function, which may be a primary codeterminant of quantitative or qualitative differences in the symptom modulation observed.

Base Sequence↗