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Biomedical subjects

H M Temin

Publications and source records attributed to H M Temin.

At least 19 recordsLinked to original sources
PubMed1998

Overview of biological effects of addition of DNA molecules to cells.

Injected DNA proceeds with certain probabilities through the following steps: degradation by serum nucleases, adsorption to cells, uptake into cells, ligation to other DNA, mutation, expression of unintegrated DNA, integration, expression of integrated DNA, and activation of or inactivation of cellular genes. The maximal probability per DNA molecule of each of these steps is estimated based on experimental results in cell culture with transfection of DNA and with infection by retroviruses. A maximum cumulative probability of having a harmful effects is calculated to be less than 10(-16) to 10(-19) per DNA molecule from a cell without activated proto-oncogenes or active viral oncogenes. The most frequent harmful effects considered are inactivation of a tumour suppressor gene and activation of a proto-oncogene. Such inactivation and activation in a cell that could give rise to cancer would increase the age-standardized incidence of cancer by a small amount. The amount of increase would differ among individuals depending upon their genotypes and their environments. Thus, the magnitude of the increase will depend upon the frequency of more sensitive individuals. The probability of an increased incidence of cancer as a possible effect of the vaccination should be compared to the number of DNA molecules to be injected per person and to the protective effects of successful HBV vaccine.

Adsorption↗
PubMed1997

In vivo study of genetically simplified bovine leukemia virus derivatives that lack tax and rex.

Genetically simplified derivatives of complex retroviruses that replicate in animal models are useful tools to study the role of the complex regulatory genes in virus infection and pathogenesis and were proposed as a novel approach toward the development of vaccines against complex retroviruses. Previously we developed genetically simple derivatives of bovine leukemia virus (BLV) that can replicate in tissue culture independently of the BLV regulatory proteins, Tax and Rex, and the RIII and GIV open reading frames (K. Boris-Lawrie and H. M. Temin, J. Virol. 69:1920-1924, 1995). These derivatives are encoded on novel, hybrid retrovirus genomes that contain transcriptional control sequences of a simple retrovirus and gag-pol or env genes of the complex BLV. The first-generation simple BLV derivatives replicate as complementary viruses (coviruses) by using separate gag-pol or env genomes, and therefore virus spread is limited to cells that are infected with both covirus genomes. Here we describe a second-generation simple BLV derivative that is encoded on a single hybrid genome. We show the virus to be replication competent by successive passage on D17 target cells and by analysis of viral RNA and proteins in the infected cells. Furthermore, we evaluate the immunogenicity and infectivity of the simple BLV derivatives in a BLV animal model. Small groups of rats were injected either with virus-producing cells or with proviral DNA. Western immunoblot analysis revealed that antibodies against the major viral antigenic determinants are induced in response to either method of introduction and that seroconversion is sustained in most of the rats for at least 6 months (the duration of the study). The magnitudes of the antiviral responses were similar in rats infected with the first-generation simple BLV coviruses, the second-generation replication-competent derivative, or wild-type BLV. Wild-type BLV typically infects peripheral blood mononuclear cells (PBMC), and the simple BLV derivatives were also found to infect PBMC as demonstrated by PCR amplification of proviral sequences and reverse transcriptase PCR amplification of viral RNA in treated rats. These results establish that simple BLV derivatives lacking tax and rex are infectious and immunogenic in rats. These viruses will be useful tools in comparative studies with BLV to evaluate the role of tax and rex in maintenance of virus load and in disease outcome.

Animals↗
PubMed1996

The recombination rate is not increased when retroviral RNA is missing an encapsidation sequence.

Retroviruses, as a result of the presence of two identical RNA molecules in their virions, recombine at a high rate. When nonhomologous RNA is present in the dimer RNA molecules, nonhomologous recombination occurs, although the rate is only 0.1% of the rate of homologous recombination. We developed a protocol to study transduction of cellular proto-oncogenes in a single cycle of retrovirus replication. The psi sequences is a cis required sequence for packaging viral RNA into viral particles. To test if the rate of nonhomologous recombination increases about 1,000 times when the psi sequence is deleted, as reported by other, we modified vectors we used previously (J. Zhang and H. M. Temin, Science 259:234-238, 1993). Our results indicated that the recombination rate did not undergo the increase of about 1,000 times when the psi sequence of a chimeric RNA was deleted.

Animals↗
PubMed1995

Genetically simpler bovine leukemia virus derivatives can replicate independently of Tax and Rex.

Retrovirus genomes have a conserved modular organization that consists of trans-acting gag, pol, and env genes that function through cis-acting sequences to replicate the RNA genome to the DNA provirus. Genetically more complex retroviruses also encode regulatory genes and cis-acting sequences that are essential for their replication. We sought to convert a more complex retrovirus into a simpler retrovirus derivative that can replicate. We constructed novel, hybrid retrovirus vectors to replicate the gag, pol, and env genes of the more complex bovine leukemia virus (BLV) in the absence of regulatory genes and cis-acting response sequences. Most of the cis-acting sequences involved in the replication and regulation of BLV were replaced by the cis-acting transcriptional control sequences of a simpler retrovirus, spleen necrosis virus. We found that the resulting hybrid BLV derivatives can replicate independently of BLV Tax and Rex and the Tax and Rex cis-acting response sequences, as measured by successive passages of virus on target cells, detection of provirus sequences, and analyses of provirus and encapsidated RNAs.

Genes, pX↗
PubMed1995

The bovine leukemia virus encapsidation signal is discontinuous and extends into the 5' end of the gag gene.

In order to define bovine leukemia virus (BLV) sequences required for efficient vector replication, a series of mutations were made in a BLV vector. Testing the replication efficiency of the vectors with a helper virus and helper plasmids allowed for separation of the mutant vectors into three groups. The replication efficiency of the first group was reduced by a factor of 7; these mutants contained deletions in the 5' end of the gag gene. The second group of mutants had replication reduced by a factor of 50 and had deletions including the 5' untranslated leader region. The third group of mutants replicated at levels comparable to those of the parental vector and contained deletions of the 3' end of the gag gene, the pol gene, and the env gene. Analysis of cytoplasmic and virion RNA levels indicated that vector RNA expression was not affected but that the vector RNA encapsidation was less efficient for group 1 and group 2 mutants. Additional mutations revealed two regions important for RNA encapsidation. The first region is a 132-nucleotide-base sequence within the gag gene (nucleotides 1015 to 1147 of the proviral DNA) and facilitates efficient RNA encapsidation in the presence of the second region. The second region includes a 147-nucleotide-base sequence downstream of the primer binding site (nucleotide 551) and near the gag gene start codon (nucleotide 698; gag begins at nucleotide 628) and is essential for RNA encapsidation. We conclude that the encapsidation signal is discontinuous; a primary signal, essential for RNA encapsidation, is largely in the untranslated leader region between the primer binding site and near the gag start codon. A secondary signal, which facilitates efficient RNA encapsidation, is in a 132-nucleotide-base region within the 5' end of the gag gene.

Animals↗
PubMed1995

Lower in vivo mutation rate of human immunodeficiency virus type 1 than that predicted from the fidelity of purified reverse transcriptase.

The level of genetic variation of human immunodeficiency virus type 1 (HIV-1), a member of the lentivirus genus of the Retroviridae family, is high relative to that of retroviruses in some other genera. The high error rates of purified HIV-1 reverse transcriptase in cell-free systems suggest an explanation for this high genetic variation. To test whether the in vivo rate of mutation during reverse transcription of HIV-1 is as high as predicted by cell-free studies, and therefore higher than that rates of mutation of retroviruses in other genera, we developed an in vivo assay for detecting forward mutations in HIV-1, using the lacZ alpha peptide gene as a reporter for mutations. This system allows the rates and types of mutations that occur during a single cycle of replication to be studied. We found that the forward mutation rate for HIV-1 was 3.4 x 10(-5) mutations per bp per cycle. Base substitution mutations predominated; G-to-A transition mutations were the most common base substitution. The in vivo mutation rates for HIV-1 are three and seven times higher than those previously reported for two other retroviruses, spleen necrosis virus and bovine leukemia virus, respectively. In contrast, our calculated in vivo mutation rate for HIV-1 is about 20-fold lower than the error rate of purified HIV-1 reverse transcriptase, with the same target sequence. This finding indicates that HIV-1 reverse transcription in vivo is not as error prone as predicted from the fidelity of purified reverse transcriptase in cell-free studies. Our data suggest that the fidelity of purified HIV-1 reverse transcriptase may not accurately reflect the level of genetic variation in a natural infection.

Animals↗
PubMed1994

High rate of mismatch extension during reverse transcription in a single round of retrovirus replication.

We made spleen necrosis virus-based retroviral vectors with mutations at the 3' end of the primer binding site region to observe the effects of terminal mismatches on retroviral replication. These vectors, when compared to a vector with the wild-type primer binding sequence, allowed us to assay the effects of the mutations on the viral titer during a single cycle of replication. The mutant vectors had titers that were comparable to the wild-type vector, indicating that reverse transcriptase has no trouble extending mismatches of as many as 3 bases under normal in vivo conditions. These results confirm and extend previous in vitro studies [Yu, H. & Goodman, M. (1992) J. Biol. Chem. 15, 10888-10896] that showed that such mismatch extension could occur in a cell-free system at high concentrations of incorrect nucleotides and in the absence of correct nucleotides. We now show that mismatch extension can occur during normal retroviral replication in cells and at normal physiological nucleotide concentrations.

Animals↗
PubMed1994

A double hairpin structure is necessary for the efficient encapsidation of spleen necrosis virus retroviral RNA.

We conducted a mutational analysis within the previously defined encapsidation sequence (E) for spleen necrosis virus (SNV), an avian retrovirus. We found that two regions are necessary for efficient SNV replication. The first region is a double hairpin structure as proposed by Konings et al. (1992, J. Virol., 66, 632-640); the second region is located downstream of the hairpins. We showed further that the double hairpin structure is required for efficient SNV RNA encapsidation. Our work is the first to demonstrate, via linker-scanning and site-directed mutagenesis, that a specific RNA secondary structure is required for the encapsidation of retroviral RNA. Analysis of a series of mutations within the E region indicates (i) that preserving the secondary structure of the two hairpins is important for efficient encapsidation and (ii) that the stem regions of the hairpins contain specific sequences critical for encapsidation. Within the hairpins, the presence of at least one of the two conserved GACG four-residue loops, but not the moderately conserved bulge sequence of the first hairpin, is crucial for function. The function of the hairpins is independent of the relative order of the two hairpins. However, the two hairpins are not redundant and are not functionally identical. Replacement of SNV double hairpin sequences with those of Moloney murine leukemia virus (M-MLV) has no detectable effect on the replication of SNV-based retrovirus vectors with reticuloendotheliosis virus strain A (REV-A) helper virus. Furthermore, replacement of the entire E sequence of SNV with that of Moloney murine sarcoma virus (M-MSV) and M-MLV results in retroviral vectors that replicate as well as SNV vectors with wild type SNV E. This result indicates that the encapsidation sequences of M-MSV/M-MLV and SNV are not virus specific and that, during packaging of SNV and MLV RNA with viral proteins from REV-A, the encapsidation sequences are recognized largely by their secondary or tertiary structures.

Animals↗
PubMed1994

One retroviral RNA is sufficient for synthesis of viral DNA.

We used previously characterized spleen necrosis virus-based retroviral vectors and helper cells to study the strand transfers that occur during the reverse-transcription phase of a single cycle of retroviral replication. The conditions used selected only for formation of an active provirus rather than for expression of multiple drug resistance markers. In nonrecombinant proviruses the minus- and plus-strand DNA primer transfers were almost completely intramolecular. However, as previously reported, recombinant proviruses contained approximately equal proportions of inter- and intramolecular minus-strand DNA primer transfers. Thus, we conclude that in the absence of recombination, one molecule of retroviral RNA is sufficient for viral DNA synthesis. Large deletions and deletions with insertions were detected primarily at a limited number of positions which appear to be hot spots for such events, the primer binding site and regions containing multiple inverted repeats. At these hot spots, the rate of deletions and deletions with insertions visible with PCR was about 10% per genome per replication cycle. Other deletions and deletions with insertions (detectable with PCR) occurred at a rate of about 0.5%/kb per replication cycle. Crossovers occurred at a rate of about 6%/kb per replication cycle under single-selection conditions. This rate is comparable to the rate that we reported previously under double-selection conditions, indicating that retroviral homologous recombination is not highly error prone. The combined rates of deletions and deletions with insertions at hot spots (10% per genome per replication cycle) and other sites (0.5%/kb per replication cycle) and the rate of crossovers (6%/kb per replication cycle) indicate that on average, full-size (10-kb) type C retroviruses undergo an additional or aberrant strand transfer about once per cycle of infection.

Animals↗
PubMed1994

Lower mutation rate of bovine leukemia virus relative to that of spleen necrosis virus.

Genetic variation of the more complex retroviruses in the human T-cell leukemia virus/bovine leukemia virus (HTLV/BLV) group is less than in some other retroviral genera. To test whether reverse transcription of HTLV/BLV group members is less error prone than that of members of other groups, we developed an assay for detecting forward mutations in BLV, similar to that developed for the simpler spleen necrosis virus (SNV). We used this system to study the rates and types of mutations that occur during a single replication cycle. We found that BLV reverse transcription is approximately two and one-half times less error prone than SNV reverse transcription (4.8 x 10(-6) versus 1.2 x 10(-5) mutation per bp per cycle, respectively). The relative numbers of all types of observed mutations (that is, base pair substitutions, frameshifts, deletions, and deletions with insertions) were similar for BLV and SNV.

Animals↗
PubMed1994

Retrovirus recombination depends on the length of sequence identity and is not error prone.

Retroviruses, as a result of the presence of two identical genomic RNA molecules in their virions, recombine at a high rate. When nonhomologous RNA is present in the dimer RNA molecules, nonhomologous recombination can occur, although the rate is very low, only 0.1% of the rate of essentially homologous recombination (J. Zhang and H. M. Temin, Science 259:234-238, 1993). We found, as is found in naturally occurring highly oncogenic retroviruses (J. Zhang and H. M. Temin, J. Virol. 67:1747-1751, 1993), that the crossovers usually occur at a short region of sequence identity. We modified the previously studied vectors to study the effect of different lengths of short regions of sequence identity in the midst of otherwise nonidentical sequences. We found that the efficiency of recombination depends on the length of this sequence identity. However, the highest rate in such molecules remained lower than for recombination between essentially homologous molecules, even when there was extensive sequence identity. Junction sequences of the recombinants indicated that retrovirus recombination is not an error-prone process as was reported for human immunodeficiency virus reverse transcriptase by using a cell-free system (J. A. Peliska and S. J. Benkovic, Science 258:1112-1118, 1992).

Base Sequence↗
PubMed1994

Copackaging of different-sized retroviral genomic RNAs: little effect on retroviral replication or recombination.

We tested the effect of copackaging retroviral vectors of different sizes on retroviral replication and recombination. Our results indicate little or no difference in replication or in the rate or pattern of the strand transfers leading to the formation of recombinant proviruses with size. The size difference of the vectors also allowed us to extend our previous analysis of the linkage of markers in the recombinant proviruses. We conclude that the observed linkage is inconsistent with the strand displacement/assimilation model of retroviral recombination.

Animals↗
PubMed1994

High rates of frameshift mutations within homo-oligomeric runs during a single cycle of retroviral replication.

Homo-oligomeric runs were inserted into a spleen necrosis virus-based retrovirus vector to determine the nature and rate of mutations within runs of 10 to 12 identical nucleotides during a single replication cycle. Clones of helper cells containing integrated copies of retroviral vectors were used to produce virus for infection of target (nonhelper) cells. Proviral sequences from target cell clones were compared with proviral sequences from helper cell clones to study mutations that occurred during a single cycle of replication. In addition to the internal region spanning the homo-oligomeric inserts, a naturally occurring run of 10 T's in the long terminal repeat (LTR) also was sequenced. Rates of mutation ranged from < 0.01 to 0.38 frameshift mutations per run per cycle for different nucleotide runs. Frameshift mutations ranged from deletions of 2 bases to additions of 5 bases; the most common mutations were +1 and -1. Frameshift mutation rates did not increase as the run length increased from 10 to 12 bases. Rates of frameshift mutation for runs of T's and A's were significantly higher than rates for runs of C's and G's, and rates for runs of pyrimidines were significantly higher than those for runs of purines. Interestingly, the vast majority of frameshift mutations in the internal region (95%) were positive, suggesting that the primer strand tends to slip backward on the template in this region. LTR runs had a significantly lower number of positive frameshift mutations than the internal runs. By analyzing the types of frameshift mutations within runs and by comparing the patterns of frameshift mutations in the 5' and 3' LTRs of individual proviruses, we conclude that the majority of mutations observed in our system occurred during minus-strand DNA synthesis of reverse transcription.

Animals↗
PubMed1993

Retrovirus variation and reverse transcription: abnormal strand transfers result in retrovirus genetic variation.

Human immunodeficiency virus variation is extensive and is based on numerous mistakes in reverse transcription. All retrovirus replication requires two strand transfers (growing point jumps) to synthesize the complete provirus. I propose that the numerous mistakes in reverse transcription are the result of this requirement for the two strand transfers needed to form the provirus.

Genetic Variation↗
PubMed1993

A proposal for a new approach to a preventive vaccine against human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) is a more complex retrovirus, coding for several accessory proteins in addition to the structural proteins (Gag, Pol, and Env) that are found in all retroviruses. More complex retroviruses have not been isolated from birds, and simpler retroviruses have not been isolated from humans. However, the proviruses of many endogenous simpler retroviruses are present in the human genome. These observations suggest that humans can mount a successful protective response against simpler retroviruses, whereas birds cannot. Thus, humans might be able to mount a successful protective response to infection with a simpler HIV-1. As a model, a simpler bovine leukemia virus which is capable of replicating has been constructed; a simpler HIV-1 could be constructed in a similar fashion. I suggest that such a simpler HIV-1 would be a safe and effective vaccine against HIV-1.

AIDS Vaccines↗