Biomedical subjects

N Mador

Publications and source records attributed to N Mador.

12 recordsLinked to original sources

PubMed1998

Herpes simplex virus type 1 latency-associated transcripts suppress viral replication and reduce immediate-early gene mRNA levels in a neuronal cell line.

During herpes simplex virus type 1 (HSV-1) latent infection in human dorsal root ganglia, limited viral transcription, which has been linked to HSV-1 reactivation ability, takes place. To study the involvement of this transcription in HSV-1 replication in neuronal cells and consequently in viral latency, we constructed stably transfected neuronal cell lines containing (i) the entire HSV-1 latency transcriptionally active DNA fragment, (ii) the same DNA sequence with deletions of the latency-associated transcript (LAT) promoters, or (iii) the DNA coding sequence of the LAT domain. Replication of HSV-1 or a LAT-negative mutant was markedly repressed in the LAT-expressing cells, a phenomenon mediated by the LATs. To study the mechanism responsible for this effect, we examined LAT influence upon expression of HSV-1 immediate-early (IE) genes ICP0, ICP4, and ICP27, by Northern blot analysis. Following infection of a LAT-expressing neuronal cell line with a LAT-negative mutant, the steady-state levels of all three IE mRNAs were reduced compared to those for control cells. Transient transfections into a neuronal cell line indicated that the LAT suppressive effect upon ICP0 mRNA was mediated directly and was not due to the LAT effect upon the ICP0 promoter. We therefore propose that the LATs may repress viral replication in neuronal cells by reducing IE gene mRNA levels and thus facilitate the establishment of HSV-1 latency in nervous tissue.

Animals↗

PubMed1998

Tissue specific distribution of the herpes simplex virus type 1 latency-associated transcripts on polyribosomes during latent infection.

Transcription of herpes simplex virus type 1 (HSV-1) during latency produces two abundant latency-associated transcripts (LATs). We have recently shown, that during HSV-1 latency in mice trigeminal ganglia (TG) LATs are bound to polyribosomes (J Virol, 1997, 71, 2897-2904). In order to study the possible role of this binding in the latency process, we now extend the polyribosomal analysis to brainstem tissues of latently infected mice, that unlike TG do not support viral reactivation. We report here that the relative amounts of the LATs associated with polyribosomes in the brainstems of mice are significantly lower than those present in TG. We therefore show that binding of the 1.5 and 2.0 kilobases LATs to polyribosomes is tissue specific and hypothesize that this association may have a role in the reactivation function of HSV-1.

Animals↗

PubMed1997

Variant mouse lymphoma cells with modified response to interferon demonstrate enhanced immunogenicity.

We have previously developed an experimental model for the xenogenization of malignant lymphoma. From highly tumorigenic S49 mouse lymphoma cells that proliferate in suspension culture (designated T-25), we selected variant clones that grew as an adherent monolayer (designated T-25-Adh) and were non-tumorigenic in syngeneic mice. Furthermore, priming of syngeneic hosts with T-25-Adh cells protected them against subsequent challenges with the tumorigenic T-25 cells. Several lines of evidence have indicated that antigens of an endogenous mouse mammary tumor virus (MMTV) are involved in the immunogenicity of T-25-Adh cells. Since interferon (IFN) is known to affect retroviral assembly and maturation on the cell membrane, we have studied the effects of IFN on endogenous MMTV-related structures, as well as on the immunogenicity of T-25-Adh cells. We observed that mouse alpha and beta interferons affect the morphogenesis of intracellular MMTV-related precursors in the immunogenic T-25-Adh cells, but not in tumorigenic T-25 cells. From T-25-Adh cells we selected variants that were either high responders or low responders to the above-mentioned interferon effect. The high-response variants were significantly more protective against tumorigenic T-25 cells than the low-response variants. Involvement of MMTV-related antigens in the immune response of the host to T-25-Adh cells was further suggested by immunoelectron-microscopical analysis, demonstrating that antisera from mice, immunized with T-25-Adh cells, interacted specifically with cell-surface MMTV budding particles. These findings indicate a novel method for xenogenization of lymphoma cells by IFN. Since endogenous retroviruses are present in all tissues of the mouse, this approach might be applicable to a wide variety of tumors.

Animals↗

PubMed1997

The abundant latency-associated transcripts of herpes simplex virus type 1 are bound to polyribosomes in cultured neuronal cells and during latent infection in mouse trigeminal ganglia.

During herpes simplex virus type 1 (HSV-1) latency, limited viral transcription takes place. This transcription has been linked to the ability of the HSV-1 genome to reactivate and consists of abundant 2.0- and 1.5-kb collinear latency-associated transcripts (LATs), spanned by minor hybridizing RNA (mLAT). The 1.5-kb LAT is derived from the 2.0-kb LAT by splicing, and both transcripts contain two large overlapping open reading frames. The molecular action mechanisms of the latency-associated gene expression are unknown, and no HSV-1 latency-encoded proteins have been convincingly demonstrated. We have cloned the entire latency-associated transcriptionally active HSV-1 DNA fragment (10.4 kb) under control of a constitutive promoter and generated a neuronal cell line (NA4) stably transfected with the viral LAT's region. NA4 cells produced the 2.0- and the 1.5-kb LATs. Northern blotting and reverse transcription-PCR analysis of RNA from NA4 cells and from trigeminal ganglia of mice latently infected with HSV-1 revealed that the two abundant LAT species were present in the polyribosomal RNA fractions. After addition of EDTA, which causes dissociation of mRNA-ribosome complexes, both LATs could be detected only in subpolyribosomal, but not in polyribosomal fractions. These results show that (i) HSV-1 LATs are bound to polyribosomes during latency in vivo, as well as in neuronal cells in vitro, and therefore might be translated, and that (ii) the NA4 cell line is a suitable tool with which to look for HSV-1 latency-encoded gene products.

Animals↗

PubMed1995

Translation efficiency of the human T-cell leukemia virus (HTLV-2) gag gene modulates the frequency of ribosomal frameshifting.

The regulation of ribosomal frameshifting during translation of the polycistronic mRNA of human T-cell leukemia virus (HTLV) was studied in a cell-free system. Translation inhibitors such as cycloheximide and puromycin antibiotics were much more effective in blocking the synthesis of the frameshift polypeptide Gag-Pro than the synthesis of the Gag product. The preferential inhibition of the frameshift polypeptide Gag-Pro by the two antibiotics was not a reflection of the different sizes of the two gene products, but rather a consequence of the effect of the inhibitors on ribosomal translation efficiencies. To further analyze the effect of translation efficiencies on ribosomal frameshifting, we compared the translation of 5'-capped RNA to noncapped RNA. The translation of 5'-capped RNA was higher, as expected. Consequently, ribosomal frameshifting producing the Gag-Pro polypeptide was enhanced when compared to the translation of noncapped RNA. Taken together these results indicate that efficiencies of translation, in conjunction with the cis regulatory genetic elements at the frameshift sites, determine the ratio of the polypeptides Gag, Gag-Pro, and Gag-Pro-Pol produced in the HTLV-infected cell. Thus, physiological changes which affect the cellular translation machinery may alter the optimal ratio of these three polyprotein products needed for virus maturation.

Cell-Free System↗

PubMed1995

Expression and splicing of the latency-associated transcripts of herpes simplex virus type 1 in neuronal and non-neuronal cell lines.

Herpes simplex virus type 1 (HSV-1) is transcriptionally active during latent infection in human peripheral sensory ganglia. This transcription has been linked to the ability of the virus to reactivate, but its potential gene products and mechanisms of action are unknown. To analyze the viral latency-related transcripts in neuronal and non-neuronal cell lines in an isolated cellular system, a 10.4 kb DNA fragment, which covers the entire viral transcriptionally active latency-associated region, was cloned under control of the constitutive cytomegalovirus promoter (pNM3). During transient transfection of a human embryonic kidney 293 cell line, pNM3 expressed high levels of the 2.0 kb latency-associated transcript (LAT) that was not polyadenylated. The 1.5 kb LAT as well as the minor hybridizing RNAs could not be identified by Northern blotting analysis. pNM3 expression was further analyzed following transfection of two neuronal, C1300 and ND7 cell lines. The 2.0 kb LAT was synthesized at high levels in these cell lines. The 1.5 kb LAT, which in vivo can be identified only during HSV-1 latent infection in tissues which facilitate reactivation, was present at very low amounts in 293 and C1300 cells using reverse transcription PCR analysis. Higher amounts of the 1.5 kb LAT were produced in ND7 cells, a neuronal cell line shown to possess neuronal-specific splicing proteins. However, the 1.5 kb LAT was present in ND7 cells in lesser amounts than produced during latent infection in peripheral sensory ganglia. This novel cellular system provides now a tool for future studies of the role of the 1.5 kb and the 2.0 kb LATs in HSV-1 latency.

Base Sequence↗

PubMed1994

Herpes simplex virus type 1 gene expression and reactivation of latent infection in the central nervous system.

Restricted gene expression takes place during latent infection of herpes simplex virus type 1 (HSV-1) in the human peripheral nervous system and has been linked with viral reactivation. The state of HSV-1 gene expression in the central nervous system (CNS) during latency is unclear and we, therefore, examined gene expression in the brainstem of experimental mice and normal humans. Only part of the transcription pattern present during latent infection in peripheral sensory ganglia (PSG) was identified in the human brainstem by in situ hybridization and Northern blot analysis for HSV-1-specific transcripts. Instead of three HSV-1 latency-associated transcripts (LATs) present in PSG and demonstrated by Northern blot analysis, only one was identified in mouse brainstem and none was detected in human brainstem. These findings might be attributed to the relatively low amounts of HSV-1-specific latency-associated RNAs in brainstem tissue. Combined with our inability to reactivate HSV-1 from explanted mouse brainstem, these findings suggest that tissue levels of latency-associated gene expression play a role in HSV-1 reactivation and have relevance to the very low incidence of HSV-1-induced CNS disease compared with peripheral mucocutaneous disease.

Animals↗

PubMed1993

Two cis-acting signals control ribosomal frameshift between human T-cell leukemia virus type II gag and pro genes.

The open reading frame of the human T-cell leukemia virus type II pro gene is arranged at a -1 position relative to the gag gene. Synthesis of the Gag-Pro fusion polyprotein is facilitated by ribosomal frameshift into the reading frame of the pro gene. Cloning of a synthetic 41-bp oligonucleotide corresponding to the gag-pro junction within a heterologous gene (nef of human immunodeficiency virus type I) and mutation analysis revealed that two cis-acting signals, an adenosine residue stretch and a dyad symmetry sequence, flanking the UAA termination codon, are required for efficient ribosomal frameshifting between gag and pro. The stability of the stem-loop structure is crucial for frameshifting.

Amino Acid Sequence↗

PubMed1989

Translation of gag, pro, and pol gene products of human T-cell leukemia virus type 2.

Sequence analysis of human T-cell leukemia proviral DNA revealed three open reading frames arranged at a -1 position relative to one another. On the basis of homology to other retroviruses, these open reading frames were assigned to the gag, pro, and pol genes. To characterize the primary protein products of these genes and their modes of synthesis, a DNA clone of human T-cell leukemia virus type 2 was transcribed and translated in vitro. Analysis of the viral proteins revealed three polyproteins with molecular masses of 58, 75, and 112 kilodaltons at relative frequencies of 100:13:0.9, respectively. These proteins were mapped on the viral genome by both internal deletions and 3'-end truncations at gag, pro, and pol, respectively. The results indicate that translation of the pol gene requires two independent frameshift events, and the readthrough frequencies at the two frameshift sites appeared to be similar.

Chromosome Mapping↗

PubMed1988

Isolation and characterization of interferon-resistant variants from S49 mouse lymphoma.

S49 mouse lymphoma cells were found to be extremely sensitive to the antiproliferative activity of interferon. These characteristics were studied to select for IFN-resistant cell variants. Some 0.6% of the parental S49 cell population were resistant to the antiproliferative and cytotoxic activities of IFN. The resistant cells were cloned and analyzed for their responses to several of the activities of IFN, namely, inhibition of encephalomyocarditis (EMC) virus, murine leukemia virus (MuLV) replications, and the induction of (2'-5') oligoadenylate synthetase. Among the clones selected some were highly resistant while others demonstrated only partial responsiveness to IFN. S49 cells demonstrate tubular structures in the cytoplasm. These structures were previously reported to be antigenically related to mouse mammary tumor virus (MMTV). We report here that IFN treatment decreases the expression of these cytoplasmic viral structures as revealed by electron microscopy. To correlate this novel antiviral activity to the more established functions of IFN we utilized the above mentioned S49 IFN-resistant variants. The anti-MMTV activity of IFN correlated with the other effects of IFN in both the highly resistant and partially responsive S49 clones. Our findings indicate that a relatively high proportion of S49 cells vary in their response to IFN. The defect in the resistant cells appears to affect a primary response to IFN which is common to its diverse activities. Furthermore, the effect of IFN on MMTV-related structures involves the usual pathway of IFN action.

2',5'-Oligoadenylate Synthetase↗

PubMed1985

Tubular structures in S49 mouse lymphoma are regulated through in vivo host-cell interaction and in vitro interferon treatment.

Malignant S49 mouse lymphoma cells that grow in suspension culture demonstrate in their cytoplasm characteristic tubular structures. These structures also appear in immunogenic, substrate-adherent variants of S49 cells that grow in culture. Upon transfer of both cell types into nude mice, the tubular structures of the adherent variants (and not the suspension-growing cells) undergo a profound alteration whereby their tubular components disappear and clusters of viruslike particles appear. These very closely resemble, on morphological grounds, precursors of B-type retroviruses. This specific in vivo interaction between the host and the S49 variant can be mimicked in culture by treatment of these cells for 24 h with 500 U/ml of mouse interferon. The suspension-growing S49 cells are unresponsive to interferon in this respect. Immunohistochemical analysis reveals that both tubular structures and the viruslike particles represent stages in the morphogenesis of mouse mammary tumor virus. A working hypothesis is advanced relating the regulation of the tubular system to the impaired tumorigenic potential of adherent S49 cells in syngeneic Balb/c hosts.

Animals↗

PubMed1984

Cell adhesiveness is related to tumorigenicity in malignant lymphoid cells.

Mouse lymphoma cells (S49) that grow in suspension culture were selected for increased tumorigenicity through continuous passages in syngeneic BALB/c mice. Developing tumors were classified as high grade malignant lymphoma, small noncleaved type. Variants were selected from these tumorigenic cells that were able to grow as a monolayer attached to their substrate, resembling, in this respect, fibroblastoid cells. Whereas the tumorigenic suspension-growing parental cells were able to induce progressive tumors with an inoculum as low as 100 cells per mouse, the adherent cells were unable to develop as tumors even at an inoculum of 1 X 10(8) cells per mouse. In addition, mice inoculated once with live adherent cells were immunized against 1 X 10(7) suspension-growing cells. Involvement of an immune response in the rejection of tumorigenic S49 cells was suggested by (a) adoptive transfer experiments in which spleen cells from immunized mice protected naive mice and (b) the appearance of antibodies in the sera of immunized syngeneic mice that specifically recognized both adherent and suspension-growing S49 cells and detected differences in [35S]methionine-labeled antigens from these cells. Antibodies raised in rabbits against adherent cells recognized three proteins of 34,000, 61,000, and 72,000 apparent molecular weight in radiolabeled adherent cell extracts that are either absent or present in small amounts in extracts of suspension-growing tumorigenic S49 cells. These findings, taken together with our previous report (Hochman, J., A. Katz, E. Levy, and S. Eshel, 1981, Nature (Lond.), 290:248-249), suggest the S49 system as a novel system for studying growth control in malignant lymphoid cells.

Animals↗