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L T Feldman

Publications and source records attributed to L T Feldman.

At least 37 records · Page 2Linked to original sources
PubMed1990

A latent, nonpathogenic HSV-1-derived vector stably expresses beta-galactosidase in mouse neurons.

A genetically engineered herpes simplex virus variant was constructed for use as a stable gene vector for neurons. To inhibit replication, the agent possessed a deletion in the immediate early gene ICP4, and to minimize reactivation from the latent state, the gene encoding the latency-associated transcript was deleted. The E. coli beta-galactosidase gene under the control of the Maloney murine leukemia virus long terminal repeat promoter was inserted into the ICP4 region. When introduced into the peripheral nervous system, this virus established latent infections and stably expressed beta-galactosidase in primary sensory neurons. Expression of beta-galactosidase over a more limited time period was observed when the latent infection was established in motor neurons of the hypoglossal nucleus. Agents of this general design have considerable potential for use as gene vectors for studies of neuronal function and correction of genetic defects affecting neurons.

Acute Disease↗
PubMed1989

Identification of the latency-associated transcript promoter by expression of rabbit beta-globin mRNA in mouse sensory nerve ganglia latently infected with a recombinant herpes simplex virus.

The herpes simplex virus type 1 latency-associated transcript (LAT) is expressed as a major species in latently infected mouse neurons. Previous sequence analysis revealed no obvious promoter elements near the 5' end of the LAT, but a TATA box and other potential promoter elements were found 700 base pairs upstream. A recombinant virus in which the rabbit beta-globin gene was inserted immediately downstream of the TATA box expressed globin mRNA and did not express the LAT. A second recombinant virus, in which this TATA box was removed, was negative for LAT expression in a latent infection. The location of the LAT promoter suggested that RNA upstream of the LAT was synthesized and degraded during latent-phase transcription. Low levels of this RNA were observed by in situ hybridization. In other experiments, RNA from a productive infection was used to detect a transcript extending from the LAT promoter to a polyadenylation signal approximately 8.5 kilobase downstream. These data suggest that the LAT may be processed from a larger transcription unit which begins distal to the TATA box 700 base pairs upstream of the LAT and extends to a polyadenylation signal almost 5 kilobases downstream of the 3' end of the LAT.

Animals↗
PubMed1988

Physical characterization of the herpes simplex virus latency-associated transcript in neurons.

RNA transfer (Northern) blot analysis was used to perform the physical characterization of the transcript expressed in murine sensory nerve ganglia latently infected with herpes simplex virus type 1. Most of this latency-associated transcript (LAT) was isolated in the poly(A)- fraction from ganglia. A smaller RNA species was also detected at less than 10% the abundance of the major one. LAT was not detected with probes from DNA outside the limits of the larger species. In situ hybridization data correlated well with Northern blot analysis; however, low levels of hybridization were seen with probes immediately outside the region of viral DNA giving positive Northern blot signals. S1 nuclease and primer extension mapping were used to locate the 5' end of the LAT 510 bases to the left of a KpnI site at 0.783 map units. The 3' end of the major latency-associated species was mapped to just within a 310-base-pair SmaI fragment located 660 to 970 base pairs to the right of the SalI site at 0.790 map units. These data were correlated with an analysis of the sequence of the DNA encoding this transcript and its possible function in the latent phase of infection.

Amino Acid Sequence↗
PubMed1987

RNA complementary to a herpesvirus alpha gene mRNA is prominent in latently infected neurons.

In initial attempts to define the molecular events responsible for the latent state of herpes simplex virus, in situ hybridization was utilized to search for virally encoded RNA transcripts in latently infected sensory neurons. The use of cloned probes representing the entire viral genome indicated that transcripts encoded within terminal repeats were present. When the alpha genes encoding ICP-0, ICP-4, and ICP-27 and the gamma 1 gene encoding VP-5 were employed, only RNA transcripts hybridizing to the ICP-0 probe were detected. In latently infected cells, the ICP-0--related transcripts were localized principally in the nucleus; this was not the case in acutely (productively) infected neurons or in neurons probed for RNA transcripts coding for actin. In Northern blotting experiments, an RNA of 2.6 kilobases was detected with the ICP-0 probe. When single-stranded DNAs from the ICP-0 region were used as probes, RNA from the strand complementary to that encoding ICP-0 messenger RNA (mRNA) was the major species detected. This RNA species may play a significant role in maintaining the latent infection.

Animals↗
PubMed1987

Effects of a temperature-sensitive mutation in the immediate-early gene of pseudorabies virus on class II and class III gene transcription.

The pseudorabies virus immediate-early protein activates transcription of both class II and class III genes. At present it is not known whether the activation of class II genes occurs through an activation of cellular factors or by a direct interaction of the immediate-early protein with factors or DNA. It is also not known whether the activation of class II and class III genes occurs by a similar or different mechanism. We utilized tsG, a temperature-sensitive mutation in the immediate-early gene of pseudorabies virus, to study the activation of viral genes transcribed by RNA polymerases II and III. Previous studies have shown that tsG inhibits wild-type adenovirus early gene transcription in coinfections at the nonpermissive temperature (L. T. Feldman and S. E. Ahlers, J. Virol. 57:13-17, 1986). Using this system of mixed infections as an assay, we obtained several results which allowed us to draw certain conclusions about the mode of action of the pseudorabies virus immediate-early protein (IEP). First, the tsG mutation inhibits the formation of new transcription complexes on class II genes, but does not affect transcription from preestablished transcription complexes formed in the presence or absence of the adenovirus E1A protein. Second, tsG does not inhibit transcription from class III genes, suggesting that the activation by IEP of class II and class III genes occurs by different mechanisms. Third, activation of transcription by the adenovirus E1A protein is not dominant to inhibition by tsG, suggesting that the temperature-sensitive IEP is involved in some physical interaction which actively inhibits transcription of viral genes.

Adenovirus Early Proteins↗
PubMed1987

Immediate-early protein of pseudorabies virus is not continuously required to reinitiate transcription of induced genes.

We examined the role of herpesvirus immediate-early proteins in inducing and maintaining transcription by using tsG, a temperature-sensitive mutant in the immediate-early gene of pseudorabies virus. Cells infected at the permissive temperature were shifted to the nonpermissive temperature. Initially, early gene transcription rates were similar at both temperatures. Early gene transcription subsequently decreased slowly over several hours. Our results suggest that immediate-early protein function is required only for the initial activation of early gene transcription and is not required for multiple reinitiation events from activated early genes.

Cell Transformation, Viral↗
PubMed1987

Binding of the pseudorabies virus immediate-early protein to single-stranded DNA.

In an attempt to correlate the ability to activate transcription with affinity for single-stranded DNA, both wild-type and temperature-sensitive pseudorabies virus immediate-early proteins were tested for the ability to bind to single-stranded DNA columns. Wild-type and temperature-sensitive immediate-early proteins bound to nonspecific single-stranded DNA columns with similar affinities at both 0 and 40 degrees C. There did not seem to be a direct correlation between the ability to activate transcription and the ability to bind to single-stranded DNA. To study further the interactions that are involved in binding to single-stranded DNA, we expressed the immediate-early protein in an Escherichia coli expression vector. In this system the expressed immediate-early protein was not phosphorylated, nor could it be complexed with mammalian cell factors. The first trp construct did not express a soluble form of the immediate-early protein, presumably due to the insoluble nature of the trp leader. We deleted a large segment of the trpE gene and found that the immediate-early fusion protein was soluble. We tested this protein for its affinity for single-stranded DNA by passage over single-stranded DNA cellulose columns. The bacterially expressed immediate-early protein bound single-stranded DNA at least as well as did the wild-type protein. Affinity for single-stranded DNA did not appear to be dependent on the phosphorylation state nor on the presence of mammalian cell factors.

Chromatography, Affinity↗
PubMed1986

Repression of adenovirus early gene expression by coinfection with a temperature-sensitive mutant in the immediate-early gene of pseudorabies virus.

Wild-type adenovirus was coinfected with a mutant temperature sensitive for the immediate-early gene of pseudorabies virus. At the nonpermissive temperature, this mutant, tsG, strongly inhibited the transcription of all adenovirus early genes, including E1A. This inhibition was not observed with wild-type pseudorabies virus coinfection or with tsG coinfection at the permissive temperature. The level of repression was dependent upon the ratio of tsG to adenovirus in the infection. The results suggest that the temperature-sensitive protein may be interacting with transcription factors on the viral DNA or with the DNA itself to inhibit adenovirus transcription.

Adenovirus Early Proteins↗
PubMed1985

Transcription of class III genes activated by viral immediate early proteins.

The adenovirus EIA and pseudorabies virus immediate early (IE) proteins induce transcription from transfected viral and nonviral genes transcribed by RNA polymerase II (class II genes). These proteins have now been shown also to activate transcription of transfected genes transcribed by RNA polymerase III (class III genes). As previously observed for class II genes, this stimulation of class III gene transcription was much greater for transfected genes than for the major endogenous cellular class III genes. Extracts made from cell lines stably expressing a transfected pseudorabies virus IE gene were 10 to 20 times more active in the in vitro transcription of exogenously added class III genes than extracts of the parental cell line. These results indicate that the E1A and IE proteins stimulate the expression of class III genes by a mechanism similar to the mechanism for stimulation of class II gene transcription by these proteins.

Adenoviridae↗
PubMed1984

The large late virus transcripts synthesized in herpesvirus suis (pseudorabies) virus-infected cells are not precursors of mRNA.

The sizes of early and late pseudorabies virus transcripts were compared to those of early and late mRNA. While the early primary transcripts were of approximately the same size as early mRNA, a large proportion of late primary transcripts was much larger than late mRNA. Furthermore, most early transcripts were transported efficiently to the cytoplasm and were relatively stable. In contrast, a large proportion of the late transcripts were retained in the nucleus and turned over rapidly. Specific retention in the nucleus of transcripts originating from some regions of the genome could be detected. These were, however, not preferentially degraded; degradation of transcripts originating from all regions of the genome, including those from which late mRNA originates, occurred. Experiments designed to determine whether part of the large transcripts are processed into mRNA revealed that most of the large late transcripts synthesized by the infected cells bear no precursor relationship to mRNA. Thus, during late phase of infection, most regions of the genome are abundantly transcribed as large RNA molecules; these are not destined to be processed into mRNA.

Animals↗
PubMed1984

Common control of the heat shock gene and early adenovirus genes: evidence for a cellular E1A-like activity.

We have employed an antiserum specific to the 70-kilodalton human heat shock protein and a cDNA clone specific to the mRNA for this protein to analyze the expression of the gene under noninducing conditions. Expression of the heat shock gene can be detected in the absence of heat induction, and this uninduced level of expression depends greatly on the particular cell type. For instance, the basal expression of the heat shock gene is at least 50 times higher in HeLa cells than in WI38 cells at both the mRNA and protein levels. We have previously shown that the inducer of transcription of the early adenovirus genes, the E1A gene product, also induces the heat shock gene, suggesting that these genes may be subject to the same regulation. We have, therefore, investigated the control of the adenovirus genes in relation to the cellular control of the heat shock gene. We find that human cells that allow a high level of uninduced expression of the heat shock gene (i.e., HeLa cells) also allow expression of the early adenovirus genes in the absence of the E1A inducer. The same is also true for the mouse F9 teratocarcinoma cell line. F9 stem cells, which constitutively express the heat shock protein, allow early adenovirus gene expression in the absence of E1A; upon differentiation induced by retinoic acid and cyclic AMP, the cells become restrictive and early viral gene expression requires the E1A gene product. Coordinately, upon differentiation there is also a loss of heat shock protein expression.

Adenoviruses, Human↗
PubMed1983

Activation of gene expression by adenovirus and herpesvirus regulatory genes acting in trans and by a cis-acting adenovirus enhancer element.

A plasmid containing the adenovirus E2 gene, a gene normally requiring E1A-mediated induction during viral infection, is expressed very poorly upon transfection into mouse L cells. If the same plasmid is transfected into 293 cells, which constitutively express the adenovirus E1A gene, or into L cells together with a plasmid containing the E1A gene, the E2 gene is expressed at higher levels. Cotransfection of the E2 plasmid with a plasmid containing the pseudorabies virus (a herpesvirus) immediate early gene results in an even higher increase in the level of E2 expression. In addition, efficient E2 expression in the absence of trans induction was obtained by inserting E1A upstream promoter sequences at the 5' or 3' end of the E2 gene, indicating that these E1A sequences possess enhancer properties. Thus the efficient expression of the E2 gene can be obtained either by a structural change in the gene itself or by a trans-acting induction.

Adenoviruses, Human↗
PubMed1983

Localization of the adenovirus E1Aa protein, a positive-acting transcriptional factor, in infected cells infected cells.

The function of the adenovirus E1Aa protein (the product of the 13S E1A mRNA) during a productive viral infection is to activate transcription of the six early viral transcription units. To study the mechanism of action of this protein, a peptide which was 13 amino acids long and had a sequence unique to the protein product of the adenovirus 13S E1A mRNA (pE1Aa) was coupled to keyhole limpet hemocyanin and used to raise an antibody in rabbits. The resulting antiserum was specific to this protein and did not react with the protein product of the 12S E1A mRNA, which shares considerable sequence with the E1Aa protein. This antiserum was used to probe for the E1Aa protein in situ by indirect immunofluorescence and in extracts of infected HeLa cells. We found that the protein was associated with large cellular structures both in the nucleus and in the cytoplasm. The nuclear form of the protein was analyzed further and was found to purify with the nuclear matrix.

Adenoviruses, Human↗
PubMed1983

Effect of adenovirus on metabolism of specific host mRNAs: transport control and specific translational discrimination.

We have studied the adenovirus-induced inhibition of host cell protein synthesis and the effect of infection on the overall metabolism of host cell mRNA during the late phase of adenovirus infection by following the fate of a number of cellular mRNAs complementary to specific cloned DNA segments. At a time in infection when the rate of total cellular protein synthesis is drastically (greater than 90%) reduced, transcription of specific cellular genes is undiminished. However, the transport of newly synthesized cellular mRNA to the cytoplasm is greatly decreased. This decreased appearance of new mRNA in the cytoplasm cannot account for the observed cessation of cell specific protein synthesis, however, since the concentration of several preexisting cellular mRNAs, including the mRNA for actin, remains unchanged throughout the course of infection. The preexisting mRNA is intact, capped, and functional as judged by its ability to direct protein synthesis in vitro in a cap-dependent fashion. The interruption in host translation appears to operate at the level of initiation directly, since we find that fewer ribosomes are associated with a given cellular mRNA after infection than before infection. Furthermore, the in vivo inhibition of cellular protein synthesis does not appear to be the result of competition with viral mRNA, since conditions which prevent the efficient initiation of translation of viral mRNA (infection with a viral mutant) do not result in the recovery of cell translation. Thus, it appears that a late adenovirus gene product directly mediates a shutoff of host protein synthesis.

Adenoviridae↗
PubMed1982

Activation of early adenovirus transcription by the herpesvirus immediate early gene: evidence for a common cellular control factor.

Adenovirus mutants carrying a defective E1A gene, such as dl312, are unable to express any of the early viral genes upon infection of HeLa cells. However, efficient expression of the other early adenovirus genes was obtained when dl312-infected HeLa cells were coinfected with pseudorabies virus, a herpesvirus. By employing a temperature-sensitive pseudorabies mutant (tsG1) it was demonstrated that the herpesvirus function responsible for the induction of adenovirus transcription was the immediate early gene, a gene required for the activation of herpesvirus early gene expression and the maintenance of early and late herpesvirus transcription. Specifically, HeLa cells coinfected with dl312 and tsG1, when shifted to the nonpermissive temperature, lost their capacity to express the early adenovirus genes. Furthermore, activation of early adenovirus gene expression in herpesvirus coinfection occurred earlier and at a higher level than in wild-type adenovirus infection. Therefore, the herpesvirus immediate early protein not only activates the early adenovirus transcription units but apparently does so more efficiently than the adenovirus E1A gene product. Because of this fact, we argue that the activation, either by the E1A protein or the herpesvirus immediately early protein, most likely occurs indirectly through interaction with a cellular protein rather than by a direct recognition of regulatory sequences at the adenovirus promoters.

Adenoviruses, Human↗