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R Swanstrom

Publications and source records attributed to R Swanstrom.

At least 73 records · Page 4Linked to original sources
PubMed1990

Rearrangements in unintegrated retroviral DNA are complex and are the result of multiple genetic determinants.

We used a replication-competent retrovirus shuttle vector based on a DNA clone of the Schmidt-Ruppin A strain of Rous sarcoma virus to characterize rearrangements in circular viral DNA. In this system, circular molecules of viral DNA present after acute infection of cultured cells were cloned as plasmids directly into bacteria. The use of a replication-competent shuttle vector permitted convenient isolation of a large number of viral DNA clones; in this study, over 1,000 clones were analyzed. The circular DNA molecules could be placed into a limited number of categories. Approximately one-third of the rescued molecules had deletions in which one boundary was very near the edge of a long terminal repeat (LTR) unit. Subtle differences in the patterns of deletions in circular DNAs with one versus two copies of the LTR sequence were observed, and differences between deletions emanating from the right and left boundaries of the LTR were seen. A virus with a missense mutation in the region of the pol gene responsible for integration and exhibiting a temperature sensitivity phenotype for replication had a marked decrease in the number of rescued molecules with LTR-associated deletions when infection was performed at the nonpermissive temperature. This result suggests that determinants in the pol gene, possibly in the integration protein, play a role in the generation of LTR-associated deletions. Sequences in a second region of the genome, probably within the viral gag gene, were also found to affect the types of circular viral DNA molecules present after infection. Sequences in this region from different strains of avian sarcoma-leukosis viruses influenced the fraction of circular molecules with LTR-associated deletions, as well as the relative proportion of circular molecules with either one or two copies of the LTR. Thus, the profile of rearrangements in unintegrated viral DNA is complex and dependent upon the nature of sequences in the gag and pol regions.

Animals↗
PubMed1989

Complete mutagenesis of the HIV-1 protease.

Retroviruses encode a protease which needs to be active for the production of infectious virions. A disabling mutation in the protease results in the production of non-infectious virus particles and examination of proteins from these mutant virions reveals unprocessed Gag and Gag-Pol precursor proteins, the substrates of the viral protease. Each amino acid of the HIV-1 protease was individually mutated using a simple mutagenesis procedure which is capable of introducing and identifying missense mutations in each residue of a protein. Phenotypic screening of these mutants in a heterologous assay system reveals three regions within the protease where multiple consecutive amino-acid residues are sensitive to mutation. These results show that random mutagenesis can be used to identify functionally important regions within a protein. Mutants with conditional phenotypes have also been identified within this collection.

Amino Acid Sequence↗
PubMed1989

Cleavage of HIV-1 gag polyprotein synthesized in vitro: sequential cleavage by the viral protease.

The virally encoded protease of human immunodeficiency virus is responsible for the processing of the gag and gag-pol polyprotein precursors to their mature polypeptides. Since correct processing of the viral polypeptides is essential for the production of infectious virus, HIV protease represents a potential target for therapeutic agents that may prove beneficial in the treatment of AIDS. In this study, full-length gag polyprotein has been synthesized in vitro to serve as a substrate for bacterially expressed HIV-1 protease. Expression of the protease in E. coli from the lac promoter was enhanced approximately five-fold by deletion of a potential hairpin loop upstream from the codon determining the amino terminus of mature protease. Extracts of induced cultures of E. coli harboring a protease-containing plasmid served as the source of protease activity. The gag polyprotein synthesized in vitro was cleaved by such lysates, producing fragments corresponding in size to p17 plus p24 and mature p24. Immunoprecipitations with monoclonal antibodies to p17 and p24 polypeptides suggest that initial cleavage of gag polyprotein occurs near the p24-p15 junction. The proteolysis was inhibited by pepstatin with an IC50 of 0.15 mM for cleavage at the p24-p15 junction and 0.02 mM for cleavage at the p17-p24 junction.

Base Sequence↗
PubMed1989

Mutational analysis of human immunodeficiency virus type 1 protease suggests functional homology with aspartic proteinases.

Processing of the retroviral gag and pol gene products is mediated by a viral protease. Bacterial expression systems have been developed which permit genetic analysis of the human immunodeficiency virus type 1 protease as measured by cleavage of the pol protein precursor. Deletion analysis of the pol reading frame locates the sequences required to encode a protein with appropriate proteolytic activity near the left end of the pol reading frame but largely outside the gag-pol overlap region, which is at the extreme left end of pol. Most missense mutations within an 11-amino-acid domain highly conserved among retroviral proteases and with sequence similarity to the active site of aspartic proteinases abolish appropriate processing, suggesting that the retrovirus proteases share a catalytic mechanism with aspartic proteinases. Substitution of the amino acids flanking the scissile bond at three of the processing sites encoded by pol demonstrates distinct sequence requirements for cleavage at these different sites. The inclusion of a charged amino acid at the processing site blocks cleavage. A subset of these substitutions also inhibits processing at the nonmutated sites.

Amino Acid Sequence↗
PubMed1988

The avian retrovirus env gene family: molecular analysis of host range and antigenic variants.

The nucleotide sequence of the env gp85-coding domain from two avian sarcoma and leukosis retrovirus isolates was determined to identify host range and antigenic determinants. The predicted amino acid sequence of gp85 from a subgroup D virus isolate of the Schmidt-Ruppin strain of Rous sarcoma virus was compared with the previously reported sequences of subgroup A, B, C, and E avian sarcoma and leukosis retroviruses. Subgroup D viruses are closely related to the subgroup B viruses but have an extended host range that includes the ability to penetrate certain mammalian cells. There are 27 amino acid differences shared between the subgroup D sequence and three subgroup B sequences. At 16 of these sites, the subgroup D sequence is identical to the sequence of one or more of the other subgroup viruses (A, C, and E). The remaining 11 sites are specific to subgroup D and show some clustering in the two large variable regions that are thought to be major determinants of host range. Biological analysis of recombinant viruses containing a dominant selectable marker confirmed the role of the gp85-coding domain in determining the host range of the subgroup D virus in the infection of mammalian cells. We also compared the sequence of the gp85-coding domain from two subgroup A viruses, Rous-associated virus type 1 and a subgroup A virus of the Schmidt-Ruppin strain of Rous sarcoma virus. The comparison revealed 24 nonconservative amino acid changes, of which 6 result in changes in potential glycosylation sites. The positions of 10 amino acid differences are coincident with the positions of 10 differences found between two subgroup B virus env gene sequences. These 10 sites identify seven domains in the sequence which may constitute determinants of type-specific antigenicity. Using a molecular recombinant, we demonstrated that type-specific neutralization of two subgroup A viruses was associated with the gp85-coding domain of the virus.

Alpharetrovirus↗
PubMed1987

Expression and processing of the AIDS virus reverse transcriptase in Escherichia coli.

The ability to express the genes of pathogenic human viruses, such as the acquired immune deficiency syndrome (AIDS) virus (also called human immunodeficiency virus) in bacterial cells affords the opportunity to study proteins that are ordinarily difficult or inconvenient to obtain in amounts sufficient for detailed analysis. A segment of the AIDS virus pol gene was expressed in Escherichia coli. Expression resulted in the appearance of reverse transcriptase activity in the bacterial cell extracts. The extracts contained two virus-related polypeptides that have the same apparent molecular weights as the two processed forms of virion-derived reverse transcriptase (p66 and p51). The formation of these two polypeptides depended on the coexpression of sequences located near the 5' end of the pol gene, a region that is thought to encode a viral protease. This bacterial system appears to generate mature forms of the AIDS virus reverse transcriptase by a proteolytic pathway equivalent to that which occurs during virus infection of human cells.

DNA Restriction Enzymes↗
PubMed1987

3'-Azido-3'-deoxythymidine inhibits the replication of avian leukosis virus.

We tested the ability of the thymidine analog 3'-azido-3'-deoxythymidine (BWA509U) to inhibit the replication of the retrovirus avian leukosis virus. Inhibition was measured with two different assays: inhibition of a single round of virus replication and inhibition of virus spread through a cell culture. With both assays, we detected inhibition of virus growth, although inhibition of a single round of virus replication required a 40-fold higher drug concentration than did inhibition of virus spread. We also detected variations in the concentrations of drug needed to inhibit virus replication in different cell types. Higher concentrations of drug were needed to inhibit virus replication in chicken embryo fibroblasts than in the continuous quail cell line QT6. Viral DNA synthesis in infected cells was shown to be inhibited in the presence of the drug. The triphosphate form of the analog acted as a competitive inhibitor of purified viral reverse transcriptase, with a Ki of 0.09 +/- 0.003 microM, and was incorporated as a chain terminator during reverse transcription of the natural viral RNA substrate in vitro.

Animals↗
PubMed1986

env genes of avian retroviruses: nucleotide sequence and molecular recombinants define host range determinants.

The env gene of avian sarcoma and leukosis retroviruses is allelic in the virus population permitting the virus to use different host cell receptors. This polymorphism has allowed the classification of these viruses into different subgroups. In order to understand further the role of viral sequences involved in determining this host range phenomenon, we constructed molecular recombinants between subgroup A, B, and E viruses and showed that the host range determinant defining subgroup specificity was located within a 1.1-kb region of the genome that included most of the coding region for the env gene product gp85. We also determined the nucleotide sequence of the region of the env gene encoding gp85 for virus isolates representing subgroup A and B viruses. We compared the predicted amino acid sequences of gp85 to themselves and to the previously published sequences of subgroup B, C, and E env genes. Based on these comparisons, we draw the following conclusions: Within the gp85 coding domain, there are four variable regions (VR-1 to VR-4) ranging in size from 9 to 52 amino acids. The variable regions are located in the same relative positions for each of the env gene alleles compared. The variable regions range in homology from 42% (A compared to B) to 57% (C compared to E) in pairwise comparisons; the flanking conserved domains are on average 95% homologous. The sequences of three different subgroup B virus isolates are highly homologous in both the conserved and variable regions. Secondary structure predictions suggest that gp85 is composed mostly of beta sheet topology. Hydrophilic loops within the variable regions may define sites of receptor interaction and binding sites for subgroup specific neutralizing antibodies.

Alleles↗
PubMed1986

Nuclease mechanism of the avian retrovirus pp32 endonuclease.

In vivo, the inferred circular retrovirus DNA precursor to the provirus contains two long terminal repeats (LTRs) in tandem. We studied the site-specific nicking of supercoiled DNA that contains tandem copies of avian retrovirus LTR DNA in vitro by using purified avian myeloblastosis virus pp32 endonuclease, Mg2+, and viral DNA substrates containing different LTR circle junction sequences. The results confirmed our previous observation that the pp32 protein generates two nicks, one in either viral DNA strand, each 2 nucleotides from the circle junction site. The specificity of nicking by pp32 was unchanged over an eight-fold range of protein concentration and with different avian retrovirus LTR circle junction substrates. These data are consistent with models which propose a role for the endonuclease in removal of two nucleotides from the LTR termini on integration of viral DNA in vivo.

Avian Leukosis Virus↗
PubMed1986

An anomalous Ty1 structure attributed to an error in reverse transcription.

We have determined the nucleotide sequence of both delta elements of a Ty1 transposon inserted near the CYC7 gene in the Saccharomyces cerevisiae CYC7-H2 mutant. The upstream delta element in this Ty1 has an unusual inverted repeat structure that may have been formed by an error during reverse transcription.

Base Composition↗
PubMed1985

A new pathway in the generation of defective retrovirus DNA.

We used a retrovirus shuttle vector to make molecular clones of circular viral DNA from infected cells. One-third of the molecules examined had deletions that started within or near the U5 domain of the long terminal repeat (LTR) region and extended a variable distance toward the gag gene. We present evidence that some of these deletions arose by cleavage of a single LTR unit, in contrast to the cleavage of tandem LTR units associated with the integration reaction. These results suggest that in the formation of defective circular DNA, the U5 domain can be recognized and cleaved in the absence of an adjacent U3 domain. The cleavage of isolated U5 domains may represent an important mechanism responsible for the generation of certain forms of both defective circular DNA and defective integrated DNA.

Animals↗
PubMed1983

Transduction of a cellular oncogene: the genesis of Rous sarcoma virus.

The oncogene of Rous sarcoma virus (v-src) arose by transduction of a cellular gene (c-src). In an effort to explore the mechanism of transduction, we have identified the splice acceptor site used in the genesis of mRNA for v-src, shown that an equivalent site is used in the splicing of mRNA for c-src, and determined the nucleotide sequence from the boundaries of homology between v-src and c-src. Our data indicate that (i) only a portion of c-src is represented within v-src, (ii) the leftward recombination between the genome of the transducing virus and c-src occurred in an intron of the cellular gene, (iii) v-src is in part a spliced version of the corresponding portion of c-src, and (iv) nucleotide sequences represented once in the genome of the transducing virus become duplicated to flank v-src. These findings indicate that the first step in transduction is probably recombination between DNA forms of the transducing viral genome and c-src and otherwise support the prevailing model for transduction by retroviruses. The carboxyl termini of the proteins encoded by v-src and c-src differ appreciably. An unidentified domain of 127 or 128 nucleotides is located at different positions in the genomes of two strains of RSV and gives evidence of being a foreign element that entered the viral genomes by genetic transposition independent of the transduction of src.

Amino Acid Sequence↗
PubMed1982

The leader sequence of the subgenomic mRNA's of Rous sarcoma virus is approximately 390 nucleotides.

The subgenomic mRNA's of Rous sarcoma virus share a common 5' leader sequence spliced from genomic RNA. We have examined the 5' terminal sequences of four Rous sarcoma virus RNAs: virion RNA and three species of intracellular mRNA which direct the synthesis of the RSV gene products. The lengths of the leaders on the RNAs were determined by the extent that they could protect cloned Rous sarcoma virus DNA fragments from S1 nuclease digestion after RNA-DNA hybridization. We found that the subgenomic mRNA's that direct the synthesis of the env and src gene products have uninterrupted spliced leader sequences of approximately 390 nucleotides, whereas virion RNA and full-length intracellular viral RNA have 5' termini homologous to the cloned viral DNA probe over at least the first 735 bases. In the accompanying manuscript we have determined the nucleotide sequence of the 5' end of the Rous sarcoma virus genome, including the candidate splice donor site identified here (Swanstrom et al., J. Virol. 41:535-541, 1982).

Avian Sarcoma Viruses↗
PubMed1982

Nucleotide sequence of the 5' noncoding region and part of the gag gene of Rous sarcoma virus.

Several functions of the retrovirus genome involve structural features in the vicinity of its 5' terminus. In an effort to further elucidate the relationship between structure and function in retrovirus RNA, we have determined the sequence of the first 1,010 nucleotides at the 5' end of the genome of Rous sarcoma virus by using the Maxam-Gilbert method to sequence suitable domains in cloned Rous sarcoma virus DNA. The results (i) locate the initiation codon for the gag gene of Rous sarcoma virus 372 nucleotides from the 5' end of viral RNA; (ii) demonstrate that this codon is preceded by three methionine codons that are apparently not used in translation; (iii) sustain previous conclusions that the principal site to which ribosomes bind on the Rous sarcoma virus genome in vitro does not contain the initiation codon for gag; (iv) permit deduction of the amino acid sequence of a viral structural protein, p19; (v) confirm the amino-terminal sequence of Pr76gag; and (vi) substantiate the identification of a splice donor site described in the accompanying manuscript (Hackett et al., J. Virol., 41:527-534, 1982).

Avian Sarcoma Viruses↗
PubMed1982

Structure of a replication intermediate in the synthesis of Rous sarcoma virus DNA in vivo.

Intermediates in the synthesis of Rous sarcoma virus DNA in vivo contain a short second strand of DNA (plus strong-stop DNA) synthesized by using the region near the 5' end of the first (minus) strand of DNA as the template. In this report, we show that the 3' end of plus strong-stop DNA is extended about 15 to 20 nucleotides beyond the 5' end of the minus-strand DNA template, probably copying a portion of the tRNATrp molecule that serves as primer for synthesis of the minus strand of DNA. The extra sequences present in plus strong-stop DNA may play a central role in the generation of the long terminal repeat present in mature forms of viral DNA.

Avian Sarcoma Viruses↗
PubMed1981

The terminal redundancy of the retrovirus genome facilitates chain elongation by reverse transcriptase.

Transcription of DNA from the RNA genome of retroviruses by reverse transcriptase involves an unusual translocation of the growing chain from the 5' end to the 3' end of the RNA template. In order to elucidate the mechanism by which this translocation occurs, we have used chain termination to analyze nascent viral DNA synthesized in vitro by avian sarcoma virus, and we have determined the nucleotide sequence of appropriate regions of viral DNA isolated from infected cells and cloned into prokaryotic vectors. Our results provide direct experimental evidence for a previously proposed model in which a short terminal redundancy in viral RNA, and a DNA copy of the redundant sequence, are used to allow the growing DNA chain to move from the 5' to the 3' end of the template. Transcription of avian sarcoma virus RNA with purified reverse transcriptase also generates an anomalous product, a hairpin DNA that arises when the initial DNA transcript folds back on itself to continue synthesis. The foldback is mediated by an inverted repeat of 5 nucleotides in the sequence of nascent DNA. Anomalous hairpin DNA is not produced by detergent-activated virions. Thus, constituents of the virions or the configuration of encapsidated viral RNA must facilitate correct transcription.

Alpharetrovirus↗
PubMed1981

Nucleotide sequence of cloned unintegrated avian sarcoma virus DNA: viral DNA contains direct and inverted repeats similar to those in transposable elements.

We have determined the nucleotide sequence of portions of two circular avian sarcoma virus (ASV) DNA molecules cloned in a prokaryotic host--vector system. The region whose sequence was determined represents the circle junction site--i.e., the site at which the ends of the unintegrated linear DNA are fused to form circular DNA. The sequence from one cloned molecule, SRA-2, shows that the circle junction site is the center of a 330-base-pair (bp) tandem direct repeat, presumably representing the fusion of the long terminal repeat (LTR) units known to be present at the ends of the linear DNA. The circle junction site is also the center of a 15-bp imperfect inverted repeat, which thus appears at the boundaries of the LTR. The structure of ASV DNA--unique coding region flanked by a direct repeat that is, in turn, terminated with a short inverted repeat--is very similar to the structure of certain transposable elements. Several features of the sequence imply that circularization to form the SRA-2 molecule occurred without loss of information from the linear DNA precursor. Circularization of another cloned viral DNA molecule, SRA-1, probably occurred by a different mechanism. The circle junction site of the SRA-1 molecule has a 63-bp deletion, which may have arisen by a mechanism that is analogous to the integration of viral DNA into the host genome. Flanking one side of the tandem direct repeat is the binding site for tRNATrp, the previously described primer for synthesis of the first strand of viral DNA. The other side of the direct repeat is flanked by a polypurine tract, A-G-G-G-A-G-G-G-G-G-A, which may represent the position of the primer for synthesis of the second strand of viral DNA. An A+T-rich region, upstream from the RNA capping site, and the sequence A-A-T-A-A-A are present within the direct repeat sequence. These sequences may serve as a promoter site and poly(A) addition signal, respectively, as proposed for other eukaryotic transcription units.

Avian Sarcoma Viruses↗