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L A Ball

Publications and source records attributed to L A Ball.

At least 37 records · Page 2Linked to original sources
PubMed1995

The termini of VSV DI particle RNAs are sufficient to signal RNA encapsidation, replication, and budding to generate infectious particles.

Infectious defective interfering (DI) particles of the negative-stranded RNA virus vesicular stomatitis virus (VSV) have been recovered from negative-sense transcripts of a plasmid that contains a full-length cDNA derived from the DI-T particle genome. In order to determine the cis-acting sequences necessary for RNA replication, encapsidation, and budding and to approximate the minimal size of RNA that can be packaged into infectious particles, we constructed a series of internal deletions in the DI cDNA to generate plasmids that could be transcribed to yield RNAs which ranged in size from 2209 nucleotides down to 102 nucleotides. All the deletion plasmids retained at least 36 nucleotides from the 5'-terminus and 51 nucleotides from the 3'-terminus of the DI genome. In cells expressing the five VSV proteins, the deleted DI RNAs were examined for their ability to be encapsidated, to replicate, and to bud to produce infectious DI particles. An RNA as small as 191 nucleotides, which contained 46 nucleotides from the 5'-end and 145 nucleotides from the 3'-end of the DI genome was encapsidated, replicated, and budded at least as efficiently as the full-length wild-type DI RNA. In contrast, a 102-nucleotide RNA that contained only the 51 nucleotides from the 5'-end of the DI RNA and its perfect 51-nucleotide complement at the 3'-end replicated poorly and failed to bud infectious DI particles. However, an RNA with an insertion of 1499-nucleotide "stuffer" sequences of non-VSV origin between the two 51-nucleotide complementary termini not only replicated but also budded infectious particles. These data show that the signals necessary for RNA encapsidation, replication, and packaging into infectious DI particles are contained within the 5'-terminal 36 nucleotides and the 3'-terminal 51 nucleotides of the DI RNA genome. Furthermore, the results show that a heterologous sequence can be replicated and packaged into infectious particles if it is flanked by the DI RNA termini.

Animals↗
PubMed1995

Primer sets developed to amplify conserved genes from filamentous ascomycetes are useful in differentiating fusarium species associated with conifers.

We examined the usefulness of primer sets designed to amplify introns within conserved genes in filamentous ascomycetes to differentiate 35 isolates representing six different species of Fusarium commonly found in association with conifer seedlings. We analyzed restriction fragment length polymorphisms (RFLP) in five amplified PCR products from each Fusarium isolate. The primers used in this study were constructed on the basis of sequence information from the H3, H4, and (beta)-tubulin genes in Neurospora crassa. Primers previously developed for the intergenic transcribed spacer region of the ribosomal DNA were also used. The degree of interspecific polymorphism observed in the PCR products from the six Fusarium species allowed differentiation by a limited number of amplifications and restriction endonuclease digestions. The level of intraspecific RFLP variation in the five PCR products was low in both Fusarium proliferatum and F. avenaceum but was high in a population sample of F. oxysporum isolates. Clustering of the 35 isolates by statistical analyses gave similar dendrograms for H3, H4, and (beta)-tubulin RFLP analysis, but a dendrogram produced by intergenic transcribed spacer analysis varied in the placement of some F. oxysporum isolates.

Journal Article↗
PubMed1995

Requirements for the self-directed replication of flock house virus RNA 1.

The larger segment (RNA 1) of the bipartite, positive-sense RNA genome of the nodavirus flock house virus encodes the viral RNA-dependent RNA polymerase. Two nonstructural viral proteins are made during the self-directed replication of this RNA: protein A (110 kDa), the translation product of RNA 1 itself, and protein B (11 kDa), the translation product of a subgenomic RNA (RNA 3) that is produced from RNA 1 during replication. To examine the roles of these proteins in RNA replication, specialized T7 transcription plasmids that contained wild-type or mutant copies of flock house virus RNA 1 cDNA were constructed and used in cells infected with the vaccinia virus-T7 RNA polymerase recombinant to make full-length transcripts that directed their own replication. Sequences in the primary transcripts that extended beyond the ends of the authentic RNA 1 sequence inhibited self-directed RNA replication, but plasmids that were constructed to minimize these terminal extensions produced primary transcripts that replicated as abundantly as authentic RNA 1. Truncation or mutation of the open reading frame for protein A eliminated self-directed replication, although the mutant RNA 1 remained a competent template for replication by wild-type protein A supplied in trans. These results showed that protein A was essential for RNA replication and that the process was not inseparably coupled to complete translation of the template. In contrast, protein B could be eliminated without inhibiting replication by mutations that disrupted the second of the two overlapping open reading frames on RNA 3. Furthermore, a mutant of RNA 1 in which the first nucleotide of the RNA 3 region was changed from G to U replicated at levels as high as those of the wild type without making either RNA 3 or protein B. However, diminishing replication levels were observed during subsequent replicative passages of RNA from both the mutants that could not make protein B. Roles for this protein that could account for the subtle phenotype of these mutants are discussed.

Animals↗
PubMed1994

Replication of the genomic RNA of a positive-strand RNA animal virus from negative-sense transcripts.

Studies of RNA replication among the positive-strand RNA animal viruses have been hindered by the apparent inability of their RNA-dependent RNA polymerases to initiate replication on the corresponding negative-sense RNAs. However, here I report that in the case of the nodavirus flock house virus (FHV), which has a bipartite positive-sense RNA genome, the viral RNA replicase can replicate a negative-sense transcript of the genome segment that encodes the viral capsid proteins. For this work, the FHV replication cycle was experimentally reconstructed in baby hamster kidney cells that were transfected with specialized transcription plasmids designed to direct the synthesis of RNAs which corresponded closely to the two genome segments of FHV. The RNA replicase encoded by the larger genome segment could utilize either the positive or the negative strand of the smaller segment as a template, and it catalyzed RNA replication to produce similar RNA products in the two situations. Surprisingly, studies of the nucleotide sequences that were required for replication showed that the 3' end of the negative-strand RNA contained only a minimal cis-acting signal. The success of these experiments will facilitate further studies of the cis- and trans-acting factors involved in the recognition and replication of negative-sense RNA in this system.

Base Sequence↗
PubMed1994

Extent of terminal complementarity modulates the balance between transcription and replication of vesicular stomatitis virus RNA.

We compared the template properties of a subgenomic RNA that contained the authentic 5' and 3' ends of the vesicular stomatitis virus genome with those of RNAs in which the wild-type termini were engineered to extend their complementarity from 8 to 51 nucleotides as seen in defective interfering RNAs. The RNA with authentic 5' and 3' ends directed abundant transcription but low replication. In contrast, RNAs with complementary termini derived from either end of the genome replicated well but transcribed poorly or not at all. These results have implications for understanding the mechanisms of RNA replication and transcription; they explain the replicative dominance of defective interfering RNAs and demonstrate that the extent of terminal complementarity rather than its exact sequence is a major determinant of whether the template predominantly directs transcription or replication.

Base Sequence↗
PubMed1994

Nodavirus RNA replication: mechanism and harnessing to vaccinia virus recombinants.

In order to harness RNA replication for the amplification of mRNAs expressed from recombinant vectors and vaccines, we constructed a VV recombinant that expressed the RNA replicase encoded in the larger genomic segment of the nodavirus FHV. When both termini of the VV-derived transcript were correct, the encoded enzyme replicated its own mRNA, and replication dominated the RNA synthetic capacity of the cell. The smaller genomic segment of FHV could also be replicated by the enzyme when supplied in trans, either by coinfection with another VV recombinant or by transfection of an appropriate plasmid. However, two requirements had to be fulfilled for replication of the smaller FHV RNA segment. The first was the prior replication of the larger genomic segment, which was interpreted as a mechanism to achieve sufficient replicase synthesis before the onset of coat protein synthesis. The second was the presence in the smaller genomic RNA of an internal region between about nucleotides 525-620. Work is in progress to elucidate the reasons for these requirements for RNA 2 replication.

Animals↗
PubMed1994

The membrane-associated and secreted forms of the respiratory syncytial virus attachment glycoprotein G are synthesized from alternative initiation codons.

Respiratory syncytial (RS) virus synthesizes two mature forms of its attachment glycoprotein G: an anchored type II integral membrane form and a smaller form that is secreted into the medium. Here we demonstrate that these two forms are synthesized as distinct primary translation products of a single species of G protein mRNA by initiation at either of two different AUGs. Mutant cDNAs which eliminated one of the other of the two AUG codons near the 5' end of the G gene open reading frame were constructed. Analysis of the proteins synthesized from these cDNAs, either by translation of transcripts in a cell-free system or in cells infected with recombinant vaccinia viruses containing either one of the mutant cDNAs, showed that elimination of either the first or the second of these AUG codons abrogated the synthesis of the membrane-anchored or the secreted form of the protein, respectively. Additionally, two unglycosylated forms of G protein which comigrated with the unglycosylated G proteins expressed by these recombinant viruses were detected in RS virus-infected cells. Since the second AUG encodes a methionine residue that lies near the middle of the signal/anchor domain, initiation at this codon resulted in a protein with a hydrophobic amino terminus. This form of the glycoprotein was efficiently secreted from cells infected with the vaccinia virus recombinant, and the amino-terminal sequence of this protein was identical to that of G protein secreted from RS virus-infected cells. Our results demonstrate that the secreted form of RS virus G protein is produced by initiation at the second AUG codon of the G open reading frame, followed by proteolytic removal of the signal/anchor domain.

Amino Acid Sequence↗
PubMed1993

cis-acting requirements for the replication of flock house virus RNA 2.

To examine the cis-acting requirements for RNA replication, a cDNA clone of flock house virus (FHV) RNA 2 was transfected into baby hamster kidney cells and transcribed to yield RNAs that had terminal extensions of different lengths or that lacked internal regions of the molecule. These RNAs were tested for their ability to be replicated by FHV replicase that was provided by cotransfection of purified FHV RNA 1. The results showed that RNA replication was inhibited by terminal extensions, particularly those at the 5' end of the RNA, despite the fact that these extensions were corrected during RNA replication. A negative-sense transcript with a 12-nucleotide 3' extension was replicated to produce a positive-sense RNA that had the correct 5' end, showing that the replicase could select its correct initiation site from within a longer template. A uridine residue at the second position of the positive strand was an important determinant of template activity. RNA molecules with large internal deletions that amounted to almost 50% of the 1,400 nucleotides of RNA 2 replicated as efficiently as full-length molecules, but only if they contained an internal region that lay between nucleotides 538 and 616. Thirty-six spontaneous deletions of RNA 2 that arose during sequential replicative passages all conserved the same internal region of the molecule. These results establish that both terminal and internal regions of FHV RNA 2 play essential roles in making the molecule a competent template for replication.

Animals↗
PubMed1993

Nonhomologous RNA recombination during negative-strand synthesis of flock house virus RNA.

During sequential replicative passages of viral RNA from the nodavirus flock house virus, spontaneous deletion of RNA sequences occurred frequently. Families of deleted RNA molecules were derived from both segments of the bipartite viral genome and found to contain single, double, or triple deletions. These deletions were attributed to template switching by the flock house virus RNA replicase, resulting in recombination between distant sequences and excision of the intervening nucleotides. From sequence analysis of the recombination junctions, we concluded that the process of template switching was influenced by both the primary sequence and the secondary structure of the RNA and that it occurred predominantly during synthesis of RNA negative strands.

Animals↗
PubMed1992

Infectious defective interfering particles of VSV from transcripts of a cDNA clone.

The generation of infectious defective interfering (DI) particles of vesicular stomatitis virus (VSV) entirely from cDNA clones is reported. Bacteriophage T7 RNA polymerase was used to direct the transcription of a complete negative-stranded genomic RNA from a cDNA clone of a VSV DI RNA in cells simultaneously expressing the five VSV proteins from separately transfected cDNA clones. The negative-stranded transcript was encapsidated with N protein, replicated by the VSV polymerase, and the replicated RNAs were assembled and budded to yield infectious DI virions. No helper VSV was required. Replication occurred at high levels and was assayed by direct biochemical means. An exact 3' terminus of the initial transcript, which was generated by autolytic cleavage using a ribozyme from hepatitis delta virus, was critical for replication.

Base Sequence↗
PubMed1992

Modulation of 2',5'-oligoadenylate synthetase in patients treated with alpha-interferon: effects of dose, schedule, and route of administration.

The interferon (IFN)-induced intracellular enzyme 2',5'-oligoadenylate (2-5A) synthetase was measured in extracts of peripheral mononuclear cells isolated from patients receiving a 300-fold range of doses of alpha interferon (IFN-alpha). The range of enzyme induction was 2.3- to 5.7-fold. The maximum fold increase varied from individual to individual as did the dose required for maximum enzyme stimulation. The magnitude and endurance of the enzyme response was a function of IFN dose and was unrelated to the duration of treatment or number of injections or to the route of administration. The enzyme assay was a more sensitive indicator of IFN administration than was measurement of the level of circulating IFN. These results substantiate the potential of a clinical 2-5A synthetase assay for monitoring IFN treatment.

2',5'-Oligoadenylate Synthetase↗
PubMed1992

Replication of nodamura virus after transfection of viral RNA into mammalian cells in culture.

Nodamura virus (NOV) was purified from the hind limbs of infected suckling mice and used as a source of the two genomic RNAs of the virus, RNA 1 and RNA 2. Upon transfection of the viral RNAs into baby hamster kidney (BHK21) cells in culture, vigorous RNA replication ensued and single-stranded RNAs 1 and 2 accumulated to reach an abundance which approximated that of the cellular rRNAs. Transient synthesis of a small subgenomic RNA (RNA 3) was also observed, and double-stranded versions of RNAs 1, 2, and 3 were detected. Three major viral proteins were synthesized in transfected cells. Protein A (about 115 kDa) and protein B (about 15 kDa) were made transiently at early times after transfection, whereas a large amount of protein alpha (43 kDa), the precursor to the two viral coat proteins, was made continuously starting later in the infectious cycle. When very low concentrations of viral RNAs were used for transfection, preferential replication of RNA 1 occurred. This result was attributed to segregation of the transfected viral RNAs to separate cells in culture and the subsequent replication and amplification of RNA 1 in cells that had received no RNA 2. Accordingly, multiple passages of the viral RNAs by transfection at the limit dilution resulted in the purification of RNA 1 free of RNA 2 and demonstrated that RNA 1 was capable of prolonged autonomous replication which was also accompanied by the continuous synthesis of RNA 3. In cells transfected with RNA 1 alone, protein alpha was not synthesized and proteins A and B were made continuously. Electron microscopic analysis of BHK21 cells 24 h after transfection with NOV RNAs 1 and 2 showed that large numbers of virus particles accumulated in the cytoplasm and formed paracrystalline arrays in some regions. Whole NOV purified from transfected BHK21 cells was infectious for suckling mice and had an electrophoretic mobility that was similar but not identical to that of NOV purified from infected mouse muscle. The high yield of NOV, its simple genetic composition, and its unusual genome strategy make this virus an attractive system for the study of viral RNA replication in animal cells.

Animals↗
PubMed1992

Cellular expression of a functional nodavirus RNA replicon from vaccinia virus vectors.

RNA replication provides a powerful means for the amplification of RNA, but to date it has been found to occur naturally only among RNA viruses. In an attempt to harness this process for the amplification of heterologous mRNAs, both an RNA replicase and its corresponding RNA templates have been expressed in functional form, using vaccinia virus-bacteriophage T7 RNA polymerase vectors. Plasmids were constructed which contained in 5'-to-3' order (i) a bacteriophage T7 promoter; (ii) a full-length cDNA encoding either the RNA replicase (RNA 1) or the coat protein (RNA 2) of flock house virus (FHV), (iii) a cDNA sequence that encoded the self-cleaving ribozyme of satellite tobacco ringspot virus, and (iv) a T7 transcriptional terminator. Both in vitro and in vivo, circular plasmids of this structure were transcribed by T7 RNA polymerase to produce RNAs with sizes that closely resembled those of the two authentic FHV genomic RNAs, RNA 1 and RNA 2. In baby hamster kidney cells that expressed authentic FHV RNA replicase, the RNA 2 (coat protein) transcripts were accurately replicated. Moreover, the RNA 1 (replicase) transcripts directed the synthesis of an enzyme that could replicate not only authentic virion-derived FHV RNA but also the plasmid-derived transcripts themselves. Under the latter conditions, replicative amplification of the RNA transcripts ensued and resulted in a high rate of synthesis of the encoded proteins. This successful expression from a DNA vector of the complex biological process of RNA replication will greatly facilitate studies of its mechanism and is a major step towards the goal of harnessing RNA replication for mRNA amplification.

Animals↗
PubMed1990

Complementation of a vesicular stomatitis virus glycoprotein G mutant with wild-type protein expressed from either a bovine papilloma virus or a vaccinia virus vector system.

Using a complementation assay, we have evaluated the potential of two eukaryotic expression systems to produce functional virus proteins. The first expression system was based on a bovine papilloma virus (BPV) eukaryotic expression vector which contained a copy of the gene for the membrane glycoprotein G of vesicular stomatitis virus (VSV). This vector was transfected into a mouse cell line, and transformed cell clones constitutively expressing VSV G protein were selected. These cell clones were then screened for their ability to support the replication of a temperature-sensitive G mutant of VSV (tsO45) at the permissive and nonpermissive temperatures. A 100-fold increase in tsO45 titer was observed in some of the G protein-producing cell lines in comparison with nonproducing cells. These results were compared with complementation by VSV G protein expressed from a second expression system utilizing a vaccinia virus (VV) recombinant which produced bacteriophage T7 RNA polymerase. T7 RNA polymerase expressed in cells infected with the vaccinia recombinant produced VSV G transcripts from a plasmid which had been transfected into these cells. This plasmid contained the VSV G gene cloned between T7 RNA polymerase initiation and termination signals. VSV G protein expressed by this system was able to complement tsO45 replication at the nonpermissive temperature, and yielded much greater levels of complemented virus than the BPV system. When calcium phosphate-mediated transfection was used to introduce the VSV G plasmid vector into cells infected with the VV recombinant, a complementation efficiency as high as 1500-fold was obtained. Using lipofectin-mediated transfection, a 15,000-fold increase in virus titer could be obtained in G protein-producing cells in contrast to nonproducing cells. At the nonpermissive temperature, yields of temperature-sensitive virus were within 10-fold of the yields obtained at the permissive temperature. Virus produced in this system was shown to be a pseudotype which contained wild-type G protein in the viral envelope but still maintained the temperature-sensitive genotype. This expression system will be used to study the extent to which the integrity of the G coding sequence of wild-type VSV might be altered in the absence of selection pressure for functional G protein during VSV replication.

Animals↗
PubMed1990

Limited expression of poliovirus by vaccinia virus recombinants due to inhibition of the vector by proteinase 2A.

A recombinant vaccinia virus was constructed that expressed poliovirus coat precursor protein P1 fused to about two-thirds of the 2A proteinase. The truncated 2A segment could be cleaved away from the P1 region by coinfecting with poliovirus type 1, 2, or 3 or with human rhinovirus 14 but not with encephalomyocarditis virus. Further cleavage of the vector-derived P1 to yield mature poliovirus capsid proteins was not observed. Attempts to isolate vaccinia virus recombinants containing portions of the poliovirus genome that encompassed the complete gene for proteinase 2A were unsuccessful, unless expression of functional 2A was abolished by insertion of a frameshift mutation. We conclude that an activity of the 2A proteinase, probably its role in translational inhibition, prevented isolation of vaccinia virus recombinants that expressed 2A.

Amino Acid Sequence↗
PubMed1989

Structure and cell surface maturation of the attachment glycoprotein of human respiratory syncytial virus in a cell line deficient in O glycosylation.

The synthesis of the extensively O-glycosylated attachment protein, G, of human respiratory syncytial virus and its expression on the cell surface were examined in a mutant Chinese hamster ovary (CHO) cell line, ldlD, which has a defect in protein O glycosylation. These cells, used in conjunction with an inhibitor of N-linked oligosaccharide synthesis, can be used to establish conditions in which no carbohydrate addition occurs or in which either N-linked or O-linked carbohydrate addition occurs exclusively. A recombinant vaccinia virus expression vector for the G protein was constructed which, as well as containing the human respiratory syncytial virus G gene, contained a portion of the cowpox virus genome that circumvents the normal host range restriction of vaccinia virus in CHO cells. The recombinant vector expressed high levels of G protein in both mutant ldlD and wild-type CHO cells. Several immature forms of the G protein were identified that contained exclusively N-linked or O-linked oligosaccharide side chains. Metabolic pulse-chase studies indicated that the pathway of maturation for the G protein proceeds from synthesis of the 32-kilodalton (kDa) polypeptide accompanied by cotranslational attachment of high-mannose N-linked sugars to form an intermediate with an apparent mass of 45 kDa. This step is followed by the Golgi-associated conversion of the N-linked sugars to the complex type and the completion of the O-linked oligosaccharides to achieve the mature 90-kDa form of G. Maturation from the 45-kDa N-linked form to the mature 90-kDa form occurred only in the presence of O-linked sugar addition, confirming that O-linked oligosaccharides constitute a significant proportion of the mass of the mature G protein. In the absence of O glycosylation, forms of G bearing galactose-deficient truncated N-linked and fully mature N-linked oligosaccharides were observed. The effects of N- and O-linked sugar addition on the transport of G to the cell surface were measured. Indirect immunofluorescence and flow cytometry showed that G protein could be expressed on the cell surface in the absence of either O glycosylation or N glycosylation. However, cell surface expression of G lacking both N- and O-linked oligosaccharides was severely depressed.

Animals↗
PubMed1988

Helper T cell recognition of respiratory syncytial virus in mice.

In this study we aimed to define the protein and viral subtype specificities of helper Th cells to respiratory syncytial virus (RSV). BALB/c mice were primed by infection with RSV, or with vaccinia viruses (VV) containing genes encoding several individual RSV proteins. Priming for Th cell memory was assayed by stimulating spleen cells in vitro with different RSV isolates and measuring RSV-specific interleukin 2 (IL-2) release by T cells into supernatants using an IL-2-dependent CTLL cell line. Splenocytes from mice primed intranasally with RSV exhibited RSV-specific Th cell memory, whereas those from unprimed mice did not. Th cell recognition was in part specific to the strain of RSV used in priming and in part cross-reactive between RSV strains. Intraperitoneal priming with RSV fusion protein-expressing VV or nucleoprotein-expressing VV induced a stronger RSV-specific Th cell response than the attachment glycoprotein-expressing VV which produced only slight Th recognition. No Th cell recognition of two non-structural proteins (1A and 1B) could be demonstrated.

Animals↗