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At least 109 records · Page 6Linked to original sources
PubMed1984

Analysis of two potential shuttle vectors containing herpes simplex virus defective DNA.

Two potential shuttle vectors which contained the identical herpes simplex virus type 1 (HSV-1) defective particle DNA (dDNA), but prokaryotic DNA of different origins, were examined for their stability when propagated in eukaryotic cells, and for their efficiency as shuttle vectors. Each chimeric molecule contained a 9.5 kilobase-pair (kb) EcoRI fragment (HSV12-7) representing a single unit of a class I HSV-1 dDNA. This dDNA was cloned into the bacteriophage lambda (lambda) vector lambda gtWES X lambda B' to create a 45.3 kb chimeric molecule (lambda gtWES::12-7), and into the plasmid vector pBR325, resulting in a 15.5 kb recombinant DNA molecule (pBR325::12-7). Each of these DNA molecules was transfected independently into African green monkey kidney cells which were then infected with wild-type HSV-1 helper virus. Both chimeric molecules were replicated and packaged into HSV-1 virions. However, regions of the lambda gtWES::12-7 chimeric DNA were rapidly deleted and rearranged, whereas the plasmid/HSV-1 DNA molecules were less rearranged. No intact lambda gtWES::12-7 DNA was recovered from HSV-1 virions as detected by infectivity of in vitro packaged DNA. However, pBR325::12-7 DNA isolated from HSV-1 virions was able to transform E. coli to ampicillin resistance. These results suggest additional considerations when designing single units of HSV-1 dDNA for use as vectors to accommodate large fragments of DNA.

Animals↗
PubMed1990

A temperature-sensitive mutant of Newcastle disease virus defective in intracellular processing of fusion protein.

A temperature-sensitive mutant (ts3) of Newcastle disease virus was physiologically characterized. All major viral structural proteins were synthesized at the permissive (37 degrees C) and nonpermissive (42 degrees C) temperatures, but the fusion (F) glycoprotein was not cleaved at 42 degrees C. In immunocytochemical electron microscopy, the F protein was abundant in the rough endoplasmic reticulum but not in cytoplasmic membrane at 42 degrees C. Noninfectious hemagglutinating virus particles containing all major structural proteins except the F protein were released at 42 degrees C from infected cells. We concluded that the defect in ts3 resides in the intracellular processing of the F protein.

Animals↗
PubMed1997

Recombinant measles viruses defective for RNA editing and V protein synthesis are viable in cultured cells.

The measles virus (MV) phosphoprotein (P) gene encodes three proteins, P, C, and V. The V protein is synthesized by pseudo-templated transcription, also designated as RNA editing: during P gene transcription one G residue is inserted at a defined position in about 50% of the mRNAs. To study the importance of sequence elements for the nontemplated G insertion, we generated recombinant MVs in which six different mutations were introduced within the region where editing occurs (3' UUUUUCCC, template strand). These viruses were then analyzed for their ability to edit their P mRNA and to produce V protein. Single U to C changes within the U stretch abolished editing. Extending the template by three C residues at the site of G insertion resulted in a less precise editing phenotype and overproduction of V. None of these mutants were impaired in their multiplication behavior when analyzed in cultured cells. However, the syncytia of a recombinant MV overproducing V protein were in general smaller and lysed 1 to 2 days later than usual.

Animals↗
PubMed1991

Hepatitis B defective virus with rearrangements in the preS gene during chronic HBV infection.

We have found a defective form of HBV2 in a HBsAg- and anti-HBe-positive patient with liver cancer. Viral deletions were identified in the preS coding region using PCR. The presence of deleted HBV forms was observed in serum, PBMC, and liver samples. After sequencing 12 clones were analyzed (subtype adr). In 9 out of 12 clones a 183-bp in-frame deletion was recorded in the preS1 region (2995 to 3177). Three out of 9 clones also yielded rearrangements of the preS2 N-terminal part. Four out of 9 showed numerous point mutations in the preS1 and preS2 sequence. In addition, 3 out of 12 clones, which did not show the 183-bp preS1 deletion were found to have small deletions and insertions in the same part of the preS1 gene. Immunological mapping using monoclonal anti-preS antibodies showed loss of preS epitopes located at the 3'-part of preS1 and the 5'-part of preS2. On the other hand, epitopes mapped to the 5'-part of preS1 and 3' of preS2 were conserved. PBMC were also tested and solely PCR showed the major form of defective HBV with preS1 183-bp deletion. However, viral deletions in the preS gene eliminated the preS2 promotor region and B- and T-cell recognition sites. In contrast to this, the preS1 binding site to hepatocytes was conserved. Therefore, such deletions would potentially lead to an impairment in viral clearance without affecting viral penetration in liver cells, possibly accounting for chronic HBV infection.

Adult↗
PubMed1991

Hepatitis B defective virus with rearrangements in the preS gene during HBV chronic infection.

We have found a defective form of hepatitis B in an anti-HBe positive chronic HBsAg carrier with liver cancer (HCC). Viral deletions were identified in the preS coding region using PCR and sequencing. The presence of deleted HBV forms was observed in serum, PBMC, and liver tissue. To conclude, rearrangements and deletions in the preS gene would potentially lead to an impairment in viral clearance without affecting viral penetration in liver cells, possibly accounting for chronic HBV infection.

Adult↗
PubMed1999

Brome mosaic virus defective RNAs generated during infection of barley plants.

Brome mosaic virus (BMV) purified from systemically infected barley leaves 8 weeks post-inoculation (p.i.) contained defective RNAs (D-RNAs). The D-RNAs were detected in total and virion RNAs extracted from infected plants at 8 weeks p.i. or later, but not before, when barley plants had been inoculated with virions either containing or lacking D-RNA. The D-RNAs were derived from genomic RNA3 by double or mainly single deletions in the 3a protein ORF, and formed a heterogeneous population. By using in vitro transcripts of D-RNA synthesized from full-length cDNA clones, the D-RNAs were shown to replicate in a helper virus-dependent manner and to be packaged into virions in barley protoplasts. Subgenomic RNA4 was produced from the D-RNA and the coat protein was also expressed. Existence of the D-RNAs together with BMV genomic RNAs in inoculated protoplasts decreased the accumulation of 3a protein but it had no apparent effect on the accumulation of BMV genomic RNA3 or the coat protein. This is the first report of naturally occurring D-RNAs generated during prolonged infection with BMV.

Base Sequence↗
PubMed1992

Replication-defective virus infection of feather buds produces a localized region of beta-galactosidase activity.

We are interested in using retroviral vectors to trace cell lineage and to introduce exogenous genes in chicken skin explant cultures. Here the LZ10 virus carrying the gene encoding beta-galactosidase was introduced to the skin explants by two different means: a) the virus was added to the media or b) the virus was microinjected into regions of the developing feather buds. Infection by microinjection led to localized expression of beta-galactosidase in the developing feather bud, while, surprisingly, infection by adding the virus to the culture media led to localized band of beta-galactosidase expression in the middle of the feather filament. The significance of this finding in skin morphogenesis and as a tool for experimental embryology is discussed.

Animals↗
PubMed1983

A frameshift mutation affecting the carboxyl terminus of the simian virus 40 large tumor antigen results in a replication- and transformation-defective virus.

We have constructed a frameshift mutation in the simian virus 40 early region using a novel method of oligonucleotide-directed mutagenesis. The mutated DNA specifies an 84,000-dalton large tumor antigen that consists of approximately equal to 75,000 daltons encoded by the wild-type reading frame and 9,000 daltons, by the alternative reading frame (wild-type large tumor antigen is approximately equal to 82,000 daltons). The frameshifted carboxyl terminus of the protein bears a strong similarity to the same region of polyoma virus middle-sized tumor antigen. We have found that the mutant DNA is unable to replicate when introduced into permissive monkey cells and incapable of transforming nonpermissive mouse cells.

Amino Acid Sequence↗
PubMed1998

Genetic analysis of poliovirus protein 3A: characterization of a non-cytopathic mutant virus defective in killing Vero cells.

A mutational and genetic analysis of the poliovirus protein 3A has led to the identification of a single amino acid mutant virus with a restrictive phenotype to form plaques in Vero cells. This mutant (I46T 3A) can be grown and amplified in HeLa cells, where virus replication takes place at wild-type levels. However, Vero cells infected with this virus cannot complete the growth cycle. I46T 3A virus has a defect in the ability to kill Vero cells, as estimated by FACS analysis of propidium iodide uptake by dead cells. Since these defects are observed under conditions where no abnormalities in the rate of synthesis and processing of the mutant polyprotein occur, the inability to induce the cytopathic effect in infected Vero cells denotes the existence of a defect in the activity of 3A, but not the level of expression of the viral genome. As a consequence of this impaired capability to generate the cytopathic effect, I46T 3A mutant viruses cannot be titrated by plaque assay in Vero cells. Only revertant viruses with the wild-type sequence arise and form lysis plaques in Vero cells. Our results suggest a role for the 3A protein (or a precursor thereof) in the virus-induced cytopathic effect. The mutant virus characterized in this work may be a useful tool to understand how poliovirus kills infected cells and carries out the final step of its life-cycle, the release of virus progeny.

Amino Acid Substitution↗
PubMed1976

Acute encephalopathy caused by defective virus infection. I. Studies of Newcastle disease virus infections in newborn and adult mice.

An acute encephalopathy caused by a defective paramyxovirus infection was studied. Newcastle disease virus (ndv), given intracerebrally, caused neurologic disease and death in mice. Infected newborn mice died by the fourth day after inoculation, and abundant amounts of virus were recovered from their brains. Infected 4-week-old mice died by the eighth day, but only minimal amounts of virus, if any, were recovered. The brains of many moribund 4-week-old mice were histologically normal and contained no NDV antigen on fluorescent antibody staining. No serum antibody to NDV was detected. These features make this infection difficult to distinguish from a metabolic encephalopathy.

Age Factors↗
PubMed1982

Bromodeoxyuridine-induced mutants of Autographa californica nuclear polyhedrosis virus defective in occlusion body formation.

Twelve temperature-sensitive (ts) and two morphology mutants of Autographa californica nuclear polyhedrosis virus were generated using the mutagen 5-bromo-2'-deoxyuridine (BrdUrd). The ts mutants grew normally at 25 degrees C but exhibited abnormal occlusion body formation when grown at 33 degrees C whereas the morphology mutants produced an abnormal cytopathic effect at 25 degrees C and 33 degrees C. None of the mutants was severely restricted for production of non-occluded virus; thus, with the eight ts mutants and the two morphology mutants incubated at 33 degrees C the drop in titre was 0.5 to 3.0 log10 from that at 25 degrees C. The mutants fell into three groups based on the amount of polyhedrin synthesized at 25 degrees C and 33 degrees C in infected cells. The eight ts mutants were combined with ts mutants constructed in a previous series of experiments and the collection was sorted into complementation groups. The collection comprised ten groups and the new mutants were sorted into seven of these. It is concluded that gene products of these complementation groups have essential roles in occlusion body formation.

Bromodeoxyuridine↗
PubMedDate not supplied

Experimental gene therapy against subcutaneously implanted glioma with a herpes simplex virus-defective vector expressing interferon-gamma.

We investigated the feasibility of local treatment or tumor vaccination with a herpes simplex virus (HSV) type 1-defective vector. The vector was engineered to express murine interferon-gamma (IFN-gamma) for experimental gene therapy against mouse glioma Rous sarcoma virus (RSV). The murine IFN-gamma gene was driven by the cytomegalovirus promoter. The helper virus (tsk) was thermosensitive; consequently, this vector could only proliferate at 31 degrees C. A high level of murine IFN-gamma expression was confirmed in vitro and in vivo by immunohistochemistry using anti-mouse IFN-gamma monoclonal antibody. This engineered vector (dvHSV/MulFN-gamma) inhibited the proliferation of mouse glioma RSV cells in vitro, and an intratumoral (i.t.) local injection of the vector caused i.t. necrosis in vivo. The immunological effect of dvHSV/MulFN-gamma was also examined in a mouse glioma RSV cell implantation model. A subcutaneous (s.c.) implant of 1 x 10(6) mouse glioma RSV cells after treatment with dvHSV/MulFN-gamma was rejected. However, the implant after treatment with an engineered HSV-defective vector containing an antisense nucleotide sequence of the murine IFN-gamma gene was not rejected. In addition, in another group of mice in which RSV cells treated with dvHSV/MulFN-gamma were implanted into a femoral (s.c.) region and nontreated RSV cells were implanted into a contralateral femoral (s.c.) region, the implanted RSV cells were rejected. The rejection of the implanted mouse glioma RSV was blocked by anti-asialo GM1, which was known to inhibit natural killer cell activity. These results revealed that the HSV-defective vector could realize a high efficiency of transfection to glioma cells through short-time treatment, and that the IFN-gamma gene transferred to the cells had the effect of tumor vaccination, which was suggested be related to natural killer cells. In conclusion, dvHSV/MulFN-gamma may be useful for the gene therapy of malignant glioma through either i.t. local injection or a practical tumor vaccination with ex vivo gene transfer.

Animals↗
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↗
PubMed1999

An immunochemical focus assay to quantify replication competent and defective viruses involved in murine acquired immunodeficiency syndrome.

Murine acquired immunodeficiency syndrome is a lymphoproliferative disease with a concurrently developing immunodeficiency. The disease is induced after injection of supernatant of a chronically infected cell line that releases a mixture of two replication competent virus classes and a replication incompetent virus species responsible for pathogenicity. An immunochemical detection assay for virus infected foci on cell monolayers has been developed. This assay allows quantification of all three types of virus.

Animals↗
PubMed1985

Nucleotide sequences responsible for generation of internally deleted Sendai virus defective interfering genomes.

The deletion points of four internally deleted defective interfering (DI) RNA species (7a, 7b, 7c, and 7d) that reside in a single Sendai virus strain were defined by nucleotide sequencing. DI RNA 7a (Mr 1.24 x 10(6)) retained the entire NP gene with the complete NP protein-coding sequence, except for the last two U residues of the polyadenylation signal, fused to an 1800-nucleotide sequence comprising 5'-terminal genome and adjacent L gene sequences. DI RNA 7b (Mr, 0.70 x 10(6)) consisted of 100 3'-terminal nucleotides fused to 1900 5'-terminal bases; the deletion point in the NP gene precedes the NP protein initiation codon. DI RNA 7c (Mr 0.55 x 10(6)) retained 420 3'-terminal and 1150 5'-terminal nucleotides. The sequence just downstream of the sequenced deletion site is M gene specific, indicating that 7c arose from at least two deletion events and that it comprises NP, M, and L gene fragments. Transcription of RNA 7c could yield an MRNA encoding a fusion protein with a 14,000 Mr (N-terminal NP sequence fused to out of frame M-specific amino acids). DI RNA 7d (Mr 0.92 x 10(6)) retained 1027 3'-terminal nucleotides fused to 1600 bases from the 5'-terminus. It has an open reading frame for a 33,000 Mr N-terminal NP protein fragment. Nucleotide sequences flanking each deletion and just downstream of the NP gene deletion site suggested that these DI genomes were generated by a copy-choice mechanism, involving polymerase jumping during replication of negative polarity virus genome templates. In this process, the termination and reinitiation of RNA synthesis would involve recognition of sequences that regulate virus genome transcription and replication.

Base Sequence↗
PubMed1994

Long-term replication of Sendai virus defective interfering particle nucleocapsids in stable helper cell lines.

An essential prerequisite for generating a stable helper cell line, which constitutively expresses functional Sendai virus RNA-dependent RNA polymerase, is the expression of all three Sendai virus nucleocapsid (NC) proteins, NP, P, and L, simulataneously. Generating a stable helper cell line was accomplished by cotransfecting cell line 293 with all three corresponding viral genes under the control of cytomegalovirus promoter-enhancer elements. Cotransfection with a dominant selectable marker enabled selection for stably transfected cells. The levels of the expressed P and NP proteins reached up to 1/10th and 1/20th of the protein levels in Sendai virus-infected cells, respectively. The Sendai virus polymerase activity of the coexpressed proteins was demonstrated by an in vivo polymerase assay. The cell clone H29 gave the strongest signal and produced DI genomes continuously for at least 3 months. This result demonstrates that it is possible to stably express adequate levels of all three viral NC proteins to form Sendai virus polymerase activity, thereby performing the replication and encapsidation of viral RNA, essential prerequisites for a helper cell line to be competent in producing recombinant viruses.

Base Sequence↗